Short beak and dwarfism syndrome among Pekin ducks: First detection, full genome sequencing, and immunohistochemical signals of novel goose parvovirus in tongue tissue.

Novel goose parvovirus (NGPV) is continuously threatening the global duck industry, as it causes short beak and dwarfism syndrome among different duck breeds. In this study, we investigated the viral pathogenesis in the tongue of affected ducks, as a new approach for deeper understanding of the syndrome. Seventy-three, 14- to 60-day-old commercial Pekin ducks were clinically examined. Thirty tissue pools of intestine and tongue (15 per tissue) were submitted for molecular identification. Clinical signs in the examined ducks were suggestive of parvovirus infection. All examined ducks had short beaks. Necrotic, swollen, and congested protruding tongues were recorded in adult ducks (37/73, 51%). Tongue protrusion without any marked congestion or swelling was observed in 20-day-old ducklings (13/73, 18%), and no tongue protrusion was observed in 15-day-old ducklings (23/73, 32%). Microscopically, the protruding tongues of adult ducks showed necrosis of the superficial epithelial layer with vacuolar degeneration. Glossitis was present in the nonprotruding tongues of young ducks, which was characterized by multifocal lymphoplasmacytic aggregates and edema in the propria submucosa. Immunohistochemical examination displayed parvovirus immunolabeling, mainly in the tongue propria submucosa. Based on polymerase chain reaction, goose parvovirus was detected in 9 out of 15 tongue sample pools (60%). Next-generation sequencing confirmed the presence of a variant goose parvovirus that is globally named NGPV and closely related to Chinese NGPV isolates. Novel insights are being gained from the study of NGPV pathogenesis in the tongue based on molecular and immunohistochemical identification.

Paternal knockdown of tRNA(cytosine-5-)-methyltransferase (Dnmt2) increases offspring susceptibility to infection in red flour beetles.

Intergenerational effects from fathers to offspring are increasingly reported from diverse organisms, but the underlying mechanisms remain speculative. Paternal trans-generational immune priming (TGIP) was demonstrated in the red flour beetle Tribolium castaneum: non-infectious bacterial exposure of fathers protects their offspring against an infectious challenge for at least two generations. Epigenetic processes, such as cytosine methylation of nucleic acids, have been proposed to enable transfer of information from fathers to offspring. Here we studied a potential role in TGIP of the Dnmt2 gene (renamed as Trdmt1 in humans), which encodes a highly conserved enzyme that methylates different RNAs, including specific cytosines of a set of tRNAs. Dnmt2 has previously been reported to be involved in intergenerational epigenetic inheritance in mice and protection against viruses in fruit flies. We first studied gene expression and found that Dnmt2 is expressed in various life history stages and tissues of T. castaneum, with high expression in the reproductive organs. RNAi-mediated knockdown of Dnmt2 in fathers was systemic, slowed down offspring larval development and increased mortality of the adult offspring upon bacterial infection. However, these effects were independent of bacterial exposure of the fathers. In conclusion, our results point towards a role of Dnmt2 for paternal effects, while elucidation of the mechanisms behind paternal TGIP needs further studies.

Detection and genetic characterization of bovine kobuvirus from calves in Egypt.

Kobuviruses are small non-enveloped RNA viruses that probably cause diarrhea in cattle and swine. Since its discovery in 2003, few studies have addressed bovine kobuvirus (BKoV; a species of Aichivirus B) infections. BKoV has been reported in Europe, Asia, and South America, suggesting a worldwide distribution. To investigate the presence of BKoV in Egypt, 36 fecal specimens from diarrheic calves in two different Egyptian provinces (Cairo and Sharkia) were screened by RT-PCR and 24 (66.7%) were found positive for BKoV. RNA from one of the positive samples (BKoV/Egy-1/KY407744) was subjected to next-generation sequencing to determine the complete BKoV genome sequence. When compared to the only recorded BKoV genome sequence (BKoV/U-1/AB084788), the studied strain showed 94 amino acid (aa) substitutions through its entire polyprotein (2463 aa), one nucleotide (nt) insertion and one nt deletion in the 2B gene and 4-nt deletions in the UTRs (2 each). Additionally, five VP1 and seven 3D sequences were obtained from other samples by using RT-PCR and Sanger sequencing. A discrepancy in the phylogenetic topography of VP1 and 3D was observed, where the Egyptian VP1 sequences were classified as a distinct cluster within the proposed lineage 1 (genotype A), which also contained strains from the UK, Brazil, and Japan. While, the 3D sequences from Cairo were related to those of Chinese strains unlike Sharkia ones that were more closer to Korean  strains. To the best of our knowledge, this is the first detection and genomic characterization of BKoV in Egypt or indeed Africa.

Molecular detection of enteric viruses from diarrheic calves in Egypt.

Neonatal calf diarrhea (NCD) is a major cause of morbidity, mortality and economic losses in the beef and dairy industries. This study was conducted to investigate the existence of enteric viruses in two Egyptian farms with a history of recurrent diarrhea. Fecal samples were collected from 25 diarrheic calves. RNA was extracted and tested by reverse transcription polymerase chain reaction (RT-PCR) for the presence of rotavirus, norovirus, astrovirus, torovirus, coronavirus and bovine viral diarrhea virus. Overall, 76 % (19/25) of samples tested positive for one or more viruses. Rota-, noro- and astroviruses were detected in 48 %, 24 % and 32 % of tested samples, respectively. About 37 % (7/19) of positive samples had two different viruses. One-month-old calves were the group most vulnerable to infections. Based on phylogenetic analysis, bovine rotaviruses were of genotypes G6 and G10, bovine noroviruses were in GIII.2, and bovine astroviruses were in the BAstV lineage 1. Astrovirus sequences showed a high level nucleotide sequence similarity with the Brazilian BAstV sequences available in GenBank. We believe this is the first report of bovine norovirus and bovine astrovirus circulating among calves in Egypt. Further epidemiological studies are recommended to investigate their presence on a wider scale, to predict their association with NCD, and to design appropriate diagnostic and control methods.

Co-circulation of paramyxo- and influenza viruses in pigeons in Egypt.

In recent years, avian influenza virus (AIV) and Newcastle disease virus (NDV) have caused large-scale outbreaks in many countries, including Egypt. The culling and vaccination strategies have failed to control both viruses in Egypt. In this study, we investigated the outbreaks of nervous manifestations and deaths in pigeons between 2013 and 2015. The H5N1 subtype of the highly pathogenic avian influenza virus and pigeon paramyxovirus-1, an antigenic variant of NDV, were found to be the cause; AIV and pigeon paramyxovirus-1 were isolated from 61.3% (19/31) and 67.8% (21/31) of tested pigeons, respectively. Co-infection with both viruses was detected in 51.6% of pigeons (16/31). The AIV sequences showed PQGEKRRKKR/GLF motif at the haemagglutinin gene cleavage site, which is typical of the highly pathogenic H5N1 subtype. The phylogenetic tree showed that the highly pathogenic avian influenza belonged to clade 2.2.1.2. The NDV sequences carried one of the three motifs, 112GKQGRL117, 112KRQKRF117 or 112RRQKRF117, at the fusion protein cleavage site and were classified as genotypes I, VI and II in NDV-class II, respectively. This indicated that different genotypes of NDV can circulate simultaneously among pigeons. Further analysis revealed the clustering of some sequences in sub-genotypes Ia and VIb.2. To the best of our knowledge, these sub-genotypes have not been previously reported from pigeons in Egypt. Our results should serve as a base for future studies on both viruses in Egypt.

Hypericum perforatum and Its Ingredients Hypericin and Pseudohypericin Demonstrate an Antiviral Activity against SARS-CoV-2.

For almost two years, the COVID-19 pandemic has constituted a major challenge to human health, particularly due to the lack of efficient antivirals to be used against the virus during routine treatment interventions. Multiple treatment options have been investigated for their potential inhibitory effect on SARS-CoV-2. Natural products, such as plant extracts, may be a promising option, as they have shown an antiviral activity against other viruses in the past. Here, a quantified extract of Hypericum perforatum was tested and found to possess a potent antiviral activity against SARS-CoV-2. The antiviral potency of the extract could be attributed to the naphtodianthrones hypericin and pseudohypericin, in contrast to other tested ingredients of the plant material, which did not show any antiviral activity. Hypericum perforatum and its main active ingredient hypericin were also effective against different SARS-CoV-2 variants (Alpha, Beta, Delta, and Omicron). Concerning its mechanism of action, evidence was obtained that Hypericum perforatum and hypericin may hold a direct virus-blocking effect against SARS-CoV-2 virus particles. Taken together, the presented data clearly emphasize the promising antiviral activity of Hypericum perforatum and its active ingredients against SARS-CoV-2 infections.

Genomic features of first bovine astrovirus detected in Egypt.

Bovine astrovirus (BAstV) is a small single-stranded RNA virus, which belongs taxonomically to genus Mamastrovirus under the family Astroviridae. The BAstV is strongly linked to neonatal diarrhea of calves. A few studies are available on BAstV, mainly from Asia, and to a lesser extent from Europe, South America, and Africa. There is only one report from Egypt, in which BAstV was found in diarrheic calves, either in single- or co-infections, based on reverse transcription polymerase chain reaction (RT-PCR) and BAstV-polymerase enzyme targeting primers. One of the samples was further subjected to genomic characterization using Illumina platform for next generation sequencing (NGS). After being processed, the returned BAstV complete genome was subjected to sequence and phylogenetic analysis in comparison to reference strains. The BAstV open reading frames (ORF1a, ORF1b, and ORF2) followed a nearly similar genetic topology, as they belonged to the same unclassified lineage, which was earlier proposed as BAstV-lineage 1, and is known to be disseminated worldwide. This close phylogenetic relationship between the study strain and other members of this lineage was further confirmed by high nucleotide and amino acid (aa) identities. Additionally, a total of 24 unique aa residues were found through the entire BAstV genome. As being the first report in Egypt, indeed Africa, we believe that this record shall be useful in either taxonomic classification or epidemiological tracking of BAstV. The status of BAstV in Egypt should be carefully investigated with possible to-be-implemented precautions for the protection of animal-raising industries. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00668-5.

Avian Paramyxovirus Type 1 in Egypt: Epidemiology, Evolutionary Perspective, and Vaccine Approach.

Avian orthoavulavirus 1, formerly known as avian paramyxovirus type-1 (APMV-1), infects more than 250 different species of birds. It causes a broad range of clinical diseases and results in devastating economic impact due to high morbidity and mortality in addition to trade restrictions. The ease of spread has allowed the virus to disseminate worldwide with subjective virulence, which depends on the virus strain and host species. The emergence of new virulent genotypes among global epizootics, including those from Egypt, illustrates the time-to-time genomic alterations that lead to simultaneous evolution of distinct APMV-1 genotypes at different geographic locations across the world. In Egypt, the Newcastle disease was firstly reported in 1947 and continued to occur, despite rigorous prophylactic vaccination, and remained a potential threat to commercial and backyard poultry production. Since 2005, many researchers have investigated the nature of APMV-1 in different outbreaks, as they found several APMV-1 genotypes circulating among various species. The unique intermingling of migratory, free-living, and domesticated birds besides the availability of frequently mobile wild birds in Egypt may facilitate the evolution power of APMV-1 in Egypt. Pigeons and waterfowls are of interest due to their inclusion in Egyptian poultry industry and their ability to spread the infection to other birds either by presence of different genotypes (as in pigeons) or by harboring a clinically silent disease (as in waterfowl). This review details (i) the genetic and pathobiologic features of APMV-1 infections in Egypt, (ii) the epidemiologic and evolutionary events in different avian species, and (iii) the vaccine applications and challenges in Egypt.

First Report of Duck Hepatitis A Virus 3 from Duckling Flocks of Egypt.

Duck hepatitis A viruses (DHAV-1, DHAV-2, and DHAV-3) are the predominant causes of duck virus hepatitis (DVH), a disease of ducklings that leads to massive morbidities, mortalities, and economic losses. As a duck-producing country, Egypt suffered lately from several attacks of DVH, despite the regular vaccination of birds. Between Spring 2016 and Summer 2018, 54 duckling flocks in the Sharkia province of Egypt were tested using the reverse-transcription PCR (RT-PCR) based on the DHAV-3D targeting primers. Of them, 27.8% (15/54) were positive. Upon retesting of positive samples using RT-PCR and duck hepatitis A virus (DHAV)-3 VP1-based primers, 33.3% (5/15) contained DHAV-3 RNA. For further analysis at the molecular level, the VP1 and the 3D genes were sequenced using the same primer sets used earlier. The phylogenetic trees confirmed that study sequences belonged to DHAV-3. However, they were displayed as a separate cluster following a geographically dependent distribution. They were also completely unrelated to the Egyptian DHAV-1-based vaccine. This was further confirmed by low nucleotide and amino acid identities in relation to this vaccine. In addition, the VP1 and 3D genes had the same phylogenetic topography. The study VP1 sequences had three unique amino acid substitutions (L59, V208 only in one strain, and C219). As far as we know, this is the first report on DHAV-3 outside Asia, particularly in Egypt. Accordingly, the vaccination strategy against DHAV should be quickly updated to avoid further dissemination of the virus. The epidemiology, pathogenicity, and evolution of DHAV-3 should be carefully monitored in Egypt.

Outbreaks of Duck Hepatitis A Virus in Egyptian Duckling Flocks.

During 2015, duck farms (n = 27) in Sharkia Province, Egypt, experienced several disease outbreaks leading to mortality and nervous manifestations. Upon necropsy, the affected ducklings showed liver lesions, such as hemorrhage or necrosis, suggestive of duck virus hepatitis (DVH). Reverse transcription-PCR (RT-PCR), on the basis of the 3D gene, found duck livers from 21 farms to be positive for duck hepatitis A virus serotype 1 (DHAV-1). All duck breeds (Pekin, Mallard, and Muscovy) were infected. The virus was isolated in embryonated chicken eggs, which showed embryonic mortality (40%-80%) within 5-7 days, stunting or dwarfing (69.6%), and necrotic liver foci (60.9%). The VP1 gene of 11 DHAV-1 strains was characterized by RT-PCR and Sanger sequencing. All study strains were clustered in a monophyletic branch within subclade B2 of Group 4 and were separated from the Egyptian vaccine strain. Several amino acid (aa) residues, such as V129, S142 (only in four strains), L181, G184, and K217, were related to virus attenuation. However, two aa residues (N193 and E205), found in virulent DHAV-1 strains, were also observed in our strains. This study confirms the circulation of DHAV-1 (subclade B2) in Lower Egypt and elucidates the phylogenetic characters of the VP1 genes, which will be useful in following the local trends of DHAV-1 infections. Further studies are indicated to determine the correlation between these mutations and the virulence of the Egyptian DHAV-1 isolates.

Brotes de virus de la hepatitis A del pato en parvadas de patitos en Egipto. Durante el año 2015, granjas de patos (n = 27) en la provincia de Sharkia en Egipto, experimentaron varios brotes de enfermedades que causaron mortalidad y manifestaciones nerviosas. Durante la necropsia, los patitos afectados mostraron lesiones hepáticas, como necrosis, hemorragia o ambas, sugestivas de la hepatitis viral del pato (con las siglas en inglés DVH). Un método de transcripción reversa y PCR (RT-PCR), sobre la base del gene 3D, encontró que los hígados de pato de 21 granjas eran positivos para el serotipo 1 del virus de la hepatitis A de pato (DHAV-1). Todas las razas de patos (Pekin, mallard y pato real) se infectaron. El virus se aisló en huevos de gallina embrionados, que mostraron una mortalidad embrionaria (40% -80%) en cinco a siete días, retraso del crecimiento o enanismo (69.6%) y focos hepáticos necróticos (60.9%). El gene VP1 de 11 cepas de DHAV-1 se caracterizó por RT-PCR y secuenciación por el método de Sanger. Todas las cepas del estudio se agruparon en una rama monofilética en el subclado B2 dentro del Grupo 4 y se separaron de la cepa de la vacuna egipcia. Varios residuos de aminoácidos, como V129, S142 (solo en cuatro cepas), L181, G184 y K217, se relacionaron con la atenuación del virus. Sin embargo, dos residuos de aminoácidos (N193 y E205), encontrados en cepas virulentas de DHAV-1, se observaron en las cepas descritas en este estudio. Este estudio confirma la circulación del virus de la hepatitis A del pato DHAV-1 (subclado B2) en el Bajo Egipto y aclara las características filogenéticas de los genes VP1, que serán útiles para seguir las tendencias locales de las infecciones por este virus. Se indican estudios adicionales para determinar la correlación entre estas mutaciones y la virulencia de los aislamientos egipcios del virus de la hepatitis A del pato.

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.