An Outbreak of Newcastle Disease Virus in the Moscow Region in the Summer of 2022.

In August 2022 on a backyard farm in the Moscow region of Russia, mortality was observed among chickens, and all 45 birds of a particular farm died or were slaughtered after the onset of symptoms within a few days. Paramyxovirus was isolated from the diseased birds. Based on the nucleotide sequences of the F and NP gene fragments, it was determined that the virus belonged to subgenotype VII.1 AAvV-1 class II. The cleavage site of the F gene 109SGGRRQKRFIG119 and T in 546 and 555 position of the NP gene were typical for the velogenic type. The genetically closest NDV isolates were found in Iran. The mean time of death of 10-day-old chicken embryos upon infection with the minimal infectious dose was 52 h, which is typical for the velogenic pathotype. The virus caused 100% death of six-week-old chickens during oral infection as well as 100% mortality of all contact chickens, including those located in remote cages, which proves the ability of the virus to spread not only by the fecal-oral route but also by the aerosol route. That demonstrates a high level of pathogenicity and contagiousness of the isolated strain for chicken. However, mice intranasally infected with high doses of the virus did not die.

Evolutionary Dynamics of Avian Influenza Viruses Isolated from Wild Birds in Moscow.

Forty-five strains of AIVs were isolated from wild aquatic birds during their autumn migration through Moscow (Russia). The aim of this work is to study the dynamics of AIV genomes in their natural habitat. Viruses were isolated from fecal sample in embryonated chicken eggs; their complete genomes were sequenced, and a phylogenetic analysis was performed. The gene segments of the same lineage persisted over the years in the absence of persistence of complete viral genomes. The genes for internal proteins of the same lineage were often maintained by the viruses over few years; however, they were typically associated with the genes of novel HA and NA subtypes. Although frequent reassortment events were observed for any pair of internal genes, there was no reassortment between HA and NA segments. The differences in the persistence of phylogenetic lineages of surface and internal proteins and the different evolutionary strategy for these two types of genes of AIVs in primary hosts are discussed.

Antigenic Architecture of the H7N2 Influenza Virus Hemagglutinin Belonging to the North American Lineage.

The North American low pathogenic H7N2 avian influenza A viruses, which lack the 220-loop in the hemagglutinin (HA), possess dual receptor specificity for avian- and human-like receptors. The purpose of this work was to determine which amino acid substitutions in HA affect viral antigenic and phenotypic properties that may be important for virus evolution. By obtaining escape mutants under the immune pressure of treatment with monoclonal antibodies, antigenically important amino acids were determined to be at positions 125, 135, 157, 160, 198, 200, and 275 (H3 numbering). These positions, except 125 and 275, surround the receptor binding site. The substitutions A135S and A135T led to the appearance of an N-glycosylation site at 133N, which reduced affinity for the avian-like receptor analog and weakened binding with tested monoclonal antibodies. Additionally, the A135S substitution is associated with the adaptation of avian viruses to mammals (cat, human, or mouse). The mutation A160V decreased virulence in mice and increased affinity for the human-type receptor analog. Conversely, substitution G198E, in combination with 157N or 160E, displayed reduced affinity for the human-type receptor analog.

Monitoring of Avian Influenza Viruses and Paramyxoviruses in Ponds of Moscow and the Moscow Region.

The ponds of the Moscow region during the autumn migration of birds are a place with large concentrations of mallard ducks, which are the main hosts of avulaviruses (avian paramyxoviruses) and influenza A viruses (IAV). The purpose of this study was the determination of the biological diversity of IAV and avulaviruses isolated from mallards in Moscow's ponds. A phylogenetic analysis of IAV was performed based on complete genome sequencing, and virus genomic reassortment in nature was studied. Almost all IAV genome segments clustered with apathogenic duck viruses according to phylogenetic analysis. The origin of the genes of Moscow isolates were different; some of them belong to European evolutionary branches, some to Asian ones. The majority of closely related viruses have been isolated in the Western Eurasian region. Much less frequently, closely related viruses have been isolated in Siberia, China, and Korea. The quantity and diversity of isolated viruses varied considerably depending on the year and have decreased since 2014, perhaps due to the increasing proportion of nesting and wintering ducks in Moscow.

Molecular Characteristics, Receptor Specificity, and Pathogenicity of Avian Influenza Viruses Isolated from Wild Ducks in Russia.

Avian influenza viruses (AIV) of wild ducks are known to be able to sporadically infect domestic birds and spread along poultry. Regular surveillance of AIV in the wild is needed to prepare for potential outbreaks. During long-year monitoring, 46 strains of AIV were isolated from gulls and mallards in Moscow ponds and completely sequenced. Amino acid positions that affect the pathogenicity of influenza viruses in different hosts were tested. The binding affinity of the virus for receptors analogs typical for different hosts and the pathogenicity of viruses for mice and chickens were investigated. Moscow isolates did not contain well-known markers of pathogenicity and/or adaptation to mammals, so as a polybasic cleavage site in HA, substitutions of 226Q and 228G amino acids in the receptor-binding region of HA, and substitutions of 627E and 701D amino acids in the PB2. The PDZ-domain ligand in the NS protein of all studied viruses contains the ESEV or ESEI sequence. Although several viruses had the N66S substitution in the PB1-F2 protein, all Moscow isolates were apathogenic for both mice and chickens. This demonstrates that the phenotypic manifestation of pathogenicity factors is not absolute but depends on the genome context.

Substitution Arg140Gly in Hemagglutinin Reduced the Virulence of Highly Pathogenic Avian Influenza Virus H7N1.

The H7 subtype of avian influenza viruses (AIV) stands out among other AIV. The H7 viruses circulate in ducks, poultry and equines and have repeatedly caused outbreaks of disease in humans. The laboratory strain A/chicken/Rostock/R0p/1934 (H7N1) (R0p), which was previously derived from the highly pathogenic strain A/FPV/Rostock/1934 (H7N1), was studied in this work to ascertain its biological property, genome stability and virulent changing mechanism. Several virus variants were obtained by serial passages in the chicken lungs. After 10 passages of this virus through the chicken lungs we obtained a much more pathogenic variant than the starting R0p. The study of intermediate passages showed a sharp increase in pathogenicity between the fifth and sixth passage. By cloning these variants, a pair of strains (R5p and R6p) was obtained, and the complete genomes of these strains were sequenced. Single amino acid substitution was revealed, namely reversion Gly140Arg in HA1. This amino acid is located at the head part of the hemagglutinin, adjacent to the receptor-binding site. In addition to the increased pathogenicity in chicken and mice, R6p differs from R5p in the shape of foci in cell culture and an increased affinity for a negatively charged receptor analogue, while maintaining a pattern of receptor-binding specificity and the pH of conformational change of HA.

Diversity and Reassortment Rate of Influenza A Viruses in Wild Ducks and Gulls.

Influenza A viruses (IAVs) evolve via point mutations and reassortment of viral gene segments. The patterns of reassortment in different host species differ considerably. We investigated the genetic diversity of IAVs in wild ducks and compared it with the viral diversity in gulls. The complete genomes of 38 IAVs of H1N1, H1N2, H3N1, H3N2, H3N6, H3N8, H4N6, H5N3, H6N2, H11N6, and H11N9 subtypes isolated from wild mallard ducks and gulls resting in a city pond in Moscow, Russia were sequenced. The analysis of phylogenetic trees showed that stable viral genotypes do not persist from year to year in ducks owing to frequent gene reassortment. For comparison, similar analyses were carried out using sequences of IAVs isolated in the same period from ducks and gulls in The Netherlands. Our results revealed a significant difference in diversity and rates of reassortment of IAVs in ducks and gulls.