In-silico evidence for enhancement of avian influenza virus H9N2 virulence by modulation of its hemagglutinin (HA) antigen function and stability during co-infection with infectious bronchitis virus in chickens.

In the last few decades, frequent incidences of avian influenza (AI) H9N2 outbreaks have caused high mortality in poultry farms resulting in colossal economic losses in several countries. In Egypt, the co-infection of H9N2 with the infectious bronchitis virus (IBV) has been observed extensively during these outbreaks. However, the pathogenicity of H9N2 in these outbreaks remained controversial. The current study reports isolation and characterization of the H9N2 virus recovered from a concurrent IBV infected broiler chicken flock in Egypt during 2011. The genomic RNA was subjected to RT-PCR amplification followed by sequencing and analysis. The deduced amino acid sequences of the eight segments of the current study H9N2 isolate were compared with those of Egyptian H9N2 viruses isolated from healthy and diseased chicken flocks from 2011 to 2013. In the phylogenetic analysis, the current study isolate was found to be closely related to the other Egyptian H9N2 viruses. Notably, no particular molecular characteristic difference was noticed among all the Egyptian H9N2 isolates from apparently healthy, diseased or co-infected with IBV chicken flocks. Nevertheless, in-silico analysis, we noted modulation of stability and motifs structure of Hemagglutinin (HA) antigen among the co-infecting H9N2 AI and the IBV and isolates from the diseased flocks. The findings suggest that the putative factor for enhancement of the H9N2 pathogenicity could be co-infection with other respiratory pathogens such as IBV that might change the HA stability and function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00688-1.

Monoclonal antibodies specific for the hemagglutinin-neuraminidase protein define neutralizing epitopes specific for Newcastle disease virus genotype 2.VII from Egypt.

BACKGROUND: Newcastle disease is a devastating disease in poultry caused by virulent Newcastle disease virus (NDV), a paramyxovirus endemic in many regions of the world despite intensive vaccination. Phylogenetic analyses reveal ongoing evolution of the predominant circulating genotype 2.VII, and the relevance of potential antigenic drift is under discussion. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Hemagglutinin-Neuraminidase (HN) spike protein, we were interested in establishing genotype-specific monoclonal antibodies (MAbs). METHODS: An HN-enriched fraction of a gradient-purified NDV genotype 2.VII was prepared and successfully employed to induce antibodies in BalbC mice that recognize conformationally intact sites reactive by haemagglutination inhibition (HI). For subsequent screening of mouse hybridoma cultures, an NDV-ELISA was established that utilizes Concanavalin A (ConA-ELISA) coupled glycoproteins proven to present conformation-dependent epitopes. RESULTS: Six out of nine selected MAbs were able to block receptor binding as demonstrated by HI activity. One MAb recognized an epitope only present in the homologue virus, while four other MAbs showed weak reactivity to selected other genotypes. On the other hand, one broadly cross-reacting MAb reacted with all genotypes tested and resembled the reactivity profile of genotype-specific polyclonal antibody preparations that point to minor antigenic differences between tested NDV genotpyes. CONCLUSIONS: These results point to the concurrent presence of variable and conserved epitopes within the HN molecule of NDV. The described protocol should help to generate MAbs against a variety of NDV strains and to enable in depth analysis of the antigenic profiles of different genotypes.

Emerging infectious bronchitis virus (IBV) in Egypt: Evidence for an evolutionary advantage of a new S1 variant with a unique gene 3ab constellation.

Infectious bronchitis virus (IBV), a gamma-coronavirus, causes infectious bronchitis (IB), a major respiratory disease of chicken. Its high mutation rate in conjunction with recombination of the RNA genome constantly creates IBV variants that are difficult to control by currently available vaccines. In this study, we addressed the question whether small-scale holdings might harbor IBV variants that serve as a reservoir for newly emerging variants. Egyptian IBV isolate EGY/NR725/2016 (NR725/16) from a small-scale broiler farm was assigned to genotype I, clade 23 (S1:GI-23), based on partial S1 gene sequences and corroborated by full genome sequencing. Analysis of the S1 gene established three subclades for historical IBV strains (S1:GI-23.1, S1:GI-23.2.1 and S1:GI-23.2.2) and confirmed NR725/16 as being part of a separate fourth subclade (S1:GI-23.3). Samples from the years 2018 and 2019 revealed that the new subclade prevails in Egypt, carrying fixed mutations within the hypervariable regions (HVR) 1-3 of the S1 protein that affect two neutralization sensitive epitopes at sites 294F, 297S and 306Y (48.2) and 329R (62.1). In addition, recombination was recognized in isolate NR 725/16, with intra-subtype mixing for the entire genes 3ab and E and inter-subtype mixing for the entire gene 6b with a close match to QX like viruses of genotype GI-19. Further analysis of gene 3ab detected the homologous gene pool to NR725/16 in samples from 2013 (3ab:C) and closely related 3ab genotypes in IBV Egyptian isolates from 2016, 2018 and 2019. These data prove a flourishing exchange between poultry holdings with a common gene pool. The continued circulation of viruses harboring genes S1:GI-23.3 and 3ab:C indicates an evolutionary advantage of this combination possibly by combining antigenic escape with modulated pathogenicity to facilitate IBV spread in the vaccinated poultry population in Egypt.

Investigation of suspected Newcastle disease (ND) outbreaks in Egypt uncovers a high virus velogenic ND virus burden in small-scale holdings and the presence of multiple pathogens.

Highly contagious Newcastle disease (ND) is associated with devastating outbreaks with highly variable clinical signs among gallinaceous birds. In this study we aimed to verify clinical ND suspicions in poultry holdings in Egypt suffering from respiratory distress and elevated mortality, comparing two groups of ND-vaccinated poultry holdings in three governorates. Besides testing for Newcastle disease virus (NDV), samples were screened for infectious bronchitis virus (IBV) and avian influenza virus (AIV) by RT-qPCR as well as by non-directed cell-culture approach on LMH-cells. Virulent NDV was confirmed only in group A (n = 16) comprising small-scale holdings. Phylogenetic analysis of the fusion protein gene of 11 NDV-positive samples obtained from this group assigned all viruses to genotype 2.VIIb and point to four different virus populations that were circulating at the same time in one governorate, indicating independent epidemiological events. In group B, comprising large commercial broiler farms (n = 10), virulent NDV was not present, although in six farms NDV vaccine-type virus (genotype 2.II) was detected. Besides, in both groups, co-infections by IBV (n = 10), AIV H9 (n = 3) and/or avian reovirus (ARV) (n = 5) and avian astrovirus (AastVs) (n = 1) could be identified. Taken together, the study confirmed clinical ND suspicion in small scale holdings, pointing to inefficient vaccination practices in this group A. However, it also highlighted that, even in an endemic situation like ND in Egypt, in cases of suspected ND vaccine failure, clinical ND suspicion has to be verified by pathotype-specific diagnostic tests. RESEARCH HIGHLIGHTS Velogenic NDV circulates in small-scale poultry holdings in Egypt. Viral transmission occurred among neighbouring farms and over long distances. Co-infections with multiple pathogens were identified. Pathotype specific diagnostic tests are essential to verify ND suspicions.