Abstract

Avian influenza is a highly dangerous viral disease that causes huge economic damage to poultry farming. Currently, highly virulent influenza virus with N8 neur- aminidase subtype is quite often detected in populations of domestic and wild birds in various countries of the world. The article provides data on complete nucleotide sequences of the neuraminidase gene of highly pathogenic avian influenza virus isolates recovered in the second half of 2020 from pathological material received from four regions of the Russian Federation. The conducted research showed that the subtype of the isolated virus was N8. According to the phylogenetic analysis, isolates of N8 virus belong to group 8C.4. During the phylogenetic analysis of the neuraminidase, we also took into account data on hemagglutinin classification, according to which H5N8 virus isolates belong to a widespread clade 2.3.4.4. Viruses of the clade were first registered in 2010 in China and they have been circulating up to now. The paper also provides data of a comparative analysis of nucleotide sequences of the studied isolates and the isolates from the international GenBank and GISAID databases, recovered in other countries from 2007 to 2020. During the analysis of the amino acid sequence of the studied isolates, no substitutions were found in the positions that affect resistance to neuraminidase inhibitors. The complete nucleotide sequences of the neuraminidase gene of the avian influenza virus subtype N8 (isolates A/domestic goose/OMSK/1521-1/2020, A/duck/Chelyabinsk/1207-1/2020, A/duck/Saratov/1578-2/2020, A/goose/Tatarstan/1730-2/2020) are published in the international GenBank and GISAID databases. Based on the analysis of the nucleotide sequences of the studied isolates, the article shows gradual evolution of the N8 subtype virus.

Highlights

  • Highly virulent influenza virus with N8 neuraminidase subtype is quite often detected in populations of domestic and wild birds in various countries of the world.The article provides data on complete nucleotide sequences of the neuraminidase gene of highly pathogenic avian influenza virus isolates recovered in the second half of 2020 from pathological material received from four regions of the Russian Federation

  • According to the phylogenetic analysis, isolates of N8 virus belong to group 8C.4

  • During the phylogenetic analysis of the neuraminidase, we took into account data on hemagglutinin classification, according to which H5N8 virus isolates belong to a widespread clade 2.3.4.4

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Summary

МАТЕРИАЛЫ И МЕТОДЫ

В работе использовали несколько систем праймеров, предназначенных для выявления РНК ВГП подтипа N8 и определения полной нуклеотидной последовательности гена нейраминидазы (NA) изолятов ВГП подтипа N8 (ЗАО «Синтол», Россия). Выделение РНК вирусов из аллантоисной жидкости куриных СПФ-эмбрионов, зараженных соответствующими изолятами, проводили с использованием набора «АмплиПрайм РИБО-сорб» (ООО «НекстБ­ ио», Россия) в соответствии с инструкцией по применению. Обратную транскрипцию с последующей амплификацией фрагментов гена NA для определения полной нуклеотидной последовательности изолятов ВГП подтипа N8 проводили в программируемых амплификаторах BioRad (Bio-Rad Laboratories, США). Синтез первой цепи комплементарной ДНК на вирусной РНК осуществляли с использованием РНКзависимой ДНК-полимеразы из вируса миелобластоза птиц (AMV Reverse Transcriptase). Амплифицированные в ПЦР фрагменты кДНК очищали с помощью набора Wizard® SV Gel and PCR Clean-Up System (Promega, США) в соответствии с инструкцией производителя. Построение филогенетического дерева проводили с помощью алгоритма NJ (в том числе с использованием метода численного ресэмплинга бутстреп) в реализации пакета MEGA, версия 7.0.26

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