Organ-specific small non-coding RNA responses in domestic (Sudani) ducks experimentally infected with highly pathogenic avian influenza virus (H5N1).

The duck represents an important reservoir of influenza viruses for transmission to other avian and mammalian hosts, including humans. The increased pathogenicity of the recently emerging clades of highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype in ducks features systemic viral spread and organ-to-organ variation in viral transcription and tissue damage. We previously reported that experimental infection of Sudani ducks (Cairina moschata) with an Egyptian HPAI (H5N1) virus (clade 2.2.1.2) features high viral replication and severe tissue damage in lung, but lower viral replication and only mild histological changes in brain. Little is known about the involvement of miRNA in organ-specific responses to H5N1 viruses in ducks, and involvement of the other classes of small noncoding RNA (sncRNA) has not been investigated so far. Following RNA sequencing, we have annotated the duck sncRNome and compared global expression changes of the four major sncRNA classes (miRNAs, piRNAs, snoRNAs, snRNAs) between duck lung and brain during a 120 h time course of infection with this HPAI strain. We find major organ-specific differences in miRNA, piRNA and snoRNA populations even before infection and substantial reprogramming of all sncRNA classes throughout infection, which was less pronounced in brain. Pathway prediction analysis of miRNA targets revealed enrichment of inflammation-, infection- and apoptosis-related pathways in lung, but enrichment of metabolism-related pathways (including tryptophan metabolism) in brain. Thus, organ-specific differences in sncRNA responses may contribute to differences in viral replication and organ damage in ducks infected with isolates from this emerging HPAI clade, and likely other strains.

Molecular characterization of equine herpes viruses type 1 and 4 among Arabian horse populations in Egypt during the period between 2021 and 2022.

Background: Equine herpesvirus type 1 (EHV-1) is a major cause of abortion and respiratory disease. Equine herpesvirus type 4 (EHV-4), on the other hand, is exclusively associated with respiratory disease in horse populations worldwide, particularly in Egypt and Arabian countries. Aim: This study aims to investigate the circulation of EHV-1 and EHV-4 in the Arabian horse population through molecular detection and genetic characterization of EHV-1 and/or EHV-4 that may threaten the stability of horse industry. Methods: A total of 80 samples including 50 nasal swabs, 10 vaginal swabs and 20 whole blood samples were collected from vaccinated and registered pure-bred Arabian adult horses from different studs in the governorates of northern Egypt (Cairo, Dakahlyia and Qalyubia) from 2021 to 2022. The collected samples were screened using consensus PCR for detection of EHVs using specific primers targeting DNA polymerase gene. The positive samples were subjected to conventional PCR for detection of EHV-1 and/or EHV-4using specific primers targeting glycoprotein (gB) gene. EHV-1 and EHV-4 amplicons were partially sequenced and phylogenetically analyzed using Sanger method. Results: Consensus PCR revealed that 48 out of 80 samples were positive for EHVs with percentage of 60%. Typing of the selected positive samples using conventional PCR showed that 29 out of 80 were positive for EHV-1 with percentage 36.25%, while 24 out of 80 samples were positive for EHV-4 with percentage 30%. Mixed infections with both viruses were detected in five samples. The amplified products were sequenced using Sanger method and submitted to GenBank under accession number OM362231MG-1 for EHV-1 strain and OM362232 MG-4 for EHV-4 strain. Sequence analysis and alignments of the amplified fragments of the EHV-1 and EHV-4 glycoprotein B (gB) gene to that of GenBank-derived reference strains revealed a high degree of similarity. According to the phylogenetic tree, the obtained sequences of EHV-1 and 4 in the current study showed homogeneity with local Egyptian and foreign EHV-1 and 4 strains and heterogeneity with EHV-2 and 5. Conclusion: The current investigation showed that molecular methods are appropriate assays for an efficient and accurate diagnosis of EHVs. Furthermore, it supports earlier research findings about the prevalence of EHV-1 and 4 in Arabian horse populations in Egypt.

The Effect of Bovine Viral Diarrhea Virus (BVDV) Strains and the Corresponding Infected-Macrophages' Supernatant on Macrophage Inflammatory Function and Lymphocyte Apoptosis.

Bovine viral diarrhea virus (BVDV) is an important viral disease of cattle that causes immune dysfunction. Macrophages are the key cells for the initiation of the innate immunity and play an important role in viral pathogenesis. In this in vitro study, we studied the effect of the supernatant of BVDV-infected macrophage on immune dysfunction. We infected bovine monocyte-derived macrophages (MDM) with high or low virulence strains of BVDV. The supernatant recovered from BVDV-infected MDM was used to examine the functional activity and surface marker expression of normal macrophages as well as lymphocyte apoptosis. Supernatants from the highly virulent 1373-infected MDM reduced phagocytosis, bactericidal activity and downregulated MHC II and CD14 expression of macrophages. Supernatants from 1373-infected MDM induced apoptosis in MDBK cells, lymphocytes or BL-3 cells. By protein electrophoresis, several protein bands were unique for high-virulence, 1373-infected MDM supernatant. There was no significant difference in the apoptosis-related cytokine mRNA (IL-1beta, IL-6 and TNF-a) of infected MDM. These data suggest that BVDV has an indirect negative effect on macrophage functions that is strain-specific. Further studies are required to determine the identity and mechanism of action of these virulence factors present in the supernatant of the infected macrophages.

Detection of equid herpesviruses among different Arabian horse populations in Egypt.

Equid herpesviruses (EHVs) threaten equine health and can cause significant economic losses to the equine industry worldwide. Different equid herpesviruses, EHV-1, EHV-2, EHV-4 and EHV5 are regularly detected among horse populations. In Egypt, monitoring is sporadic but EHV-1 or EHV-4 have been reported to circulate in the horse population. However, there is a lack of reports related to infection and health status of horses, likely due to the absence of regular diagnostic procedures. In the current study, the circulation of four infectious equid herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5) among different Arabian horse populations and donkeys residing the same farm was monitored. Different samples were collected and DNA was extracted and subjected to quantitative (q)-PCR to detect the four equid herpesviruses using specific primers and probes. Antibody titres against EHV-1 and EHV-4 were tested using virus neutralization test and type-specific ELISA. The results showed that EHV-1, EHV-2, EHV-4 and EHV-5 are endemic and can be a continuous threat for horses in the absence of vaccination programs and frequent virus reactivation. There is an urgent need for introduction of active regular surveillance measures to investigate the presence of different equid herpesviruses, and other equine viral pathogens, in various horse populations around Egypt and to establish a standardized cataloguing of equine health status.

Phylogeny of bovine norovirus in Egypt based on VP2 gene.

Bovine norovirus (BNoV) has emerged as a viral pathogen that causes a gastrointestinal illness and diarrhea in cattle. Despite its worldwide distribution, very little information is known about BNoV in Africa. In this study, BNoV was detected in 27.6% (8/29) of tested fecal materials, collected from sporadic cases of diarrheic calves, using the reverse transcription-polymerase chain reaction (RT-PCR) and primers that target RNA dependent RNA polymerase gene. Additionally, one primer pair was designed to flank the BNoV-VP2 (small capsid protein) gene for molecular analysis. Study VP2 sequences were phylogenetically-related to BNoV-GIII.2 (Newbury2-like) genotype, which is highly prevalent all over the world. However, they were separated within the cluster and one strain (41FR) grouped with recombinant GIII.P1/GIII.2 strains. Compared to reference VP2 sequences, 14 amino acid substitution mutations were found to be unique to our strains. The study confirms that BNoV is currently circulating among diarrheic calves of Egypt and also characterizes its ORF3 (VP2) genetically. The status of BNoV should be continuously evaluated in Egypt for effective prevention and control.

Pathogenicity of Highly Pathogenic Avian Influenza Virus H5N1 in Naturally Infected Poultry in Egypt.

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been endemic in Egypt since 2006, and there is increasing concern for its potential to become highly transmissible among humans. Infection by HPAIV H5N1 has been described in experimentally challenged birds. However, the pathogenicity of the H5N1 isolated in Egypt has never been reported in naturally infected chickens and ducks. Here we report a 2013 outbreak of HPAIV H5N1 in commercial poultry farms and backyards in Sharkia Province, Egypt. The main symptoms were ecchymosis on the shanks and feet, cyanosis of the comb and wattles, subcutaneous edema of the head and neck for chickens, and nervous signs (torticollis) for ducks. Within 48-72 hrs of the onset of illness, the average mortality rates were 22.8-30% and 28.5-40% in vaccinated chickens and non-vaccinated ducks, respectively. Tissue samples of chickens and ducks were collected for analyses with cross-section immunohistochemistry and real-time RT-PCR for specific viral RNA transcripts. While viral RNA was detected in nearly all tissues and sera collected, viral nucleoprotein was detected almost ubiquitously in all tissues, including testis. Interestingly, viral antigen was also observed in endothelial cells of most organs in chickens, and clearly detected in the trachea and brain in particular. Viral nucleoprotein was also detected in mononuclear cells of various organs, especially pulmonary tissue. We performed phylogenetic analyses and compared the genomic sequences of the hemagglutinin (HA) and nonstructural proteins (NS) among the isolated viruses, the HPAIV circulated in Egypt in the past and currently, and some available vaccine strains. Further analysis of deduced amino acids of both HA and NS1 revealed that our isolates carried molecular determinants of HPAIV, including the multibasic amino acids (PQGERRRK/KR*GLF) in the cleavage site in HA and glutamate at position 92 (D92E) in NS1. This is the first report of the pathogenicity of the HPAIVH5N1 strain currently circulating in naturally infected poultry in Egypt, which may provide unique insights into the viral pathogenesis in HPAIV-infected chickens and ducks.