Monitoring SARS-CoV-2 infection in different animal species and human in Egypt during 2020-2021.

Coronaviruses cause respiratory and intestinal infections in animals and humans. By the end of 2019, there was an epidemic of novel coronavirus (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses have a highly mutable genome that makes them genetically and phenotypically modifiable with a potential transmission to new host species. Based on current sequence databases, all human coronaviruses have animal origins, so animals have important roles in virus spillover to humans. The aim of this study is to investigate the role of different animal species in the epidemiology of SARS-CoV-2 in Egypt. A pan-coronaviruses RT-PCR has been used for detection of possible coronaviruses infection in different species including bats, humans, birds, and dogs in Egypt during the period of November 2020 till June 2021. Ninety-two samples (46 from Rousettus aegyptiacus bats, 10 from human, 26 from wild birds, and 10 from dogs) were screened for SARS-CoV-2. Our results revealed that only human samples were SARS-CoV-2 positive for SARS-CoV-2 while all other animal and bird samples were negative. To recapitulate, our results suggest that animals may not actively transmit SARS-CoV-2 among people in Egypt during the current COVID-19 pandemic. Further structural surveillance and follow up screening for SARS-CoV-2 among domestic and wild animal populations in Egypt is crucially needed.

A multiplex real-time reverse transcription polymerase chain reaction assay for differentiation of classical and variant II strains of avian infectious bronchitis virus.

Identification of avian infectious bronchitis virus (IBV) genotypes is essential for controlling infectious bronchitis (IB) disease, because vaccines that differ from the circulating strains might not provide efficient cross-protection. In Egypt, IBV strain typing is a difficult process, due to the widespread distribution of four genotype lineages (GI-13, GI-23, GI-1, and GI-16), which may contribute to IBV vaccination failure. In this study, we developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (mRT-qPCR) assay that targets highly conserved areas of the S1 gene in order to detect classical (G1) and Egyptian variant II (G23) strains in allantoic fluids and clinical samples. The viral genotyping technique was assessed using commercially available vaccines as well as local strains, and 16 field isolates were tested to investigate its clinical applicability. The assay was found to be specific for the detection of classical and VAR II strains and did not detect the VAR I strain or other avian pathogens such as Newcastle disease virus, avian influenza virus (H9N2 and H5N8), or infectious bursal disease virus. The results also showed that 28 out of 41 samples tested positive for IBV utilizing rt-qRT-PCR targeting the N gene and that 26 out of the 28 positive samples were genotyped by mRT-qPCR targeting the S1 gene, whereas the remaining two samples that were not genotyped were VAR 1 (4/91) and VAR I (793/B). Interestingly, the testing could identify combined infections in one sample, indicating a mixed infection with both genotypes. The real-time RT-PCR assay could detect viral RNA at concentrations as low as 102 EID50 /ml for both classical and variant II. This assay is rapid, specific, and sensitive. It appears to be a valuable tool for regular disease monitoring that can be used to differentiate as well as identify viruses.

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.

Characterization of highly pathogenic avian influenza H5N8 virus from Egyptian domestic waterfowl in 2017.

In 2016, the highly pathogenic avian influenza (HPAI) H5N8 virus was detected in wild birds for the first time in Egypt. In the present study, we identified the HPAI virus H5N8 of clade 2.3.4.4 from domestic waterfowl in Egypt, suggesting its transmission to the domestic poultry from the migratory birds. Based on partial haemagglutinin gene sequence, this virus has a close genetic relationship with subtype H5N8 viruses circulating in Asia and Europe. Pathologically, H5N8 virus in hybrid duck induced nervous signs accompanied by encephalomalacia, haemorrhages, nonsuppurative encephalitis and nonsuppurative vasculitis. The granular layer of cerebellum showed multifocal areas of hydropic degeneration and the Purkinje cell neurons were necrotized or lost. Additionally, the lung, kidney and spleen were congested, and necrotizing pancreatitis was also observed. The co-circulation of both HPAI H5N1 and H5N8 subtypes with the low pathogenic avian influenza H9N2 subtype complicate the control of avian influenza in Egypt with the possibility of emergence of new reassortant viruses. Therefore, continuous monitoring with implementation of strict control measures is required. Research highlights HPAI H5N8 virus clade 2.3.4.4 was detected in domestic ducks and geese in Egypt in 2017. Phylogenetically, the virus was closely related to HPAI H5N8 viruses identified in Asia and Europe Nonsuppurative encephalitis was widely observed in HPAI H5N8 virus-infected ducks. Degeneration of the cerebellar granular layer was found in most of the brain tissues examined.

Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus.

A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology.

Genetic characterization of a rare H12N3 avian influenza virus isolated from a green-winged teal in Japan.

This study reports on the genetic characterization of an avian influenza virus, subtype H12N3, isolated from an Eurasian green-winged teal (Anas crecca) in Japan in 2009. The entire genome sequence of the isolate was analyzed, and phylogenetic analyses were conducted to characterize the evolutionary history of the isolate. Phylogenetic analysis of the hemagglutinin and neuraminidase genes indicated that the virus belonged to the Eurasian-like avian lineage. Molecular dating indicated that this H12 virus is likely a multiple reassortant influenza A virus. This is the first reported characterization of influenza A virus subtype H12N3 isolated in Japan and these data contribute to the accumulation of knowledge on the genetic diversity and generation of novel influenza A viruses.

Molecular characterization of chicken anemia virus circulating in chicken flocks in egypt.

Introduction. Although many previous studies reported detection of chicken anemia virus (CAV) in Egypt since 1990, genomic characterization of this circulating CAV has not been published. In the present study, four nucleotide sequences of detected CAV were genetically characterized. Methods. These nucleotide sequences were obtained from commercial chicken flocks in two different locations of Egypt during 2010. The target region for sequencing was 675 bp nucleotide of partial coding region of VP1 protein. The nucleotide and deduced amino acid sequences of the detected CAV were aligned and compared to worldwide CAV isolates including commonly used vaccine strains. Phylogenetic analysis of these sequences was also carried out. Results. Our results showed that all the Egyptian CAV sequences were grouped in one group with viruses from diverse geographic regions. This group is characterized by amino acids profile (75)I, (97)L, (139)Q, and (144)Q in VP1. The phylogenetic and amino acid analyses of deduced amino acid indicated that the detected CAV sequences differ from CAV vaccine strains. Conclusion. This is the first report that describes molecular characterization of circulating CAV in Egypt. The study showed that the detected CAV, in Egypt are field viruses and unrelated to vaccine strains.

Effects of pesticide compounds (chlorothalonil and mancozeb) and benzo[a]pyrene mixture on aryl hydrocarbon receptor, p53 and ubiquitin gene expression levels in haemocytes of soft-shell clams (Mya arenaria).

The aim of this study is to investigate the effects of the pesticides/polycyclic aromatic hydrocarbon mixture on aryl hydrocarbon receptor (AhR), p53 and ubiquitin mRNA level in haemocytes of Mya arenaria exposed to a mixture of chlorothalonil, mancozeb and benzo[a]pyrene (BaP) for 48 and 72 h. AhR, p53 and ubiquitin gene expression levels were quantified using quantitative Real-time PCR. For robust and accurate quantification of transcripts, suitable housekeeping genes were selected from four sets of ribosomal and elongation factors transcripts previously sequenced from Mya arenaria using geNorm open source software. Quantitative Real-time PCR data exhibited a significantly high expression of AhR after 72 h of exposure (P ≤ 0.05). p53 gene expression seems to be up-regulated by the mixture after 48 h, however not significantly; but the level of p53 mRNA is down-regulated by the xenobiotics between 48 and 72 h after exposure. This study postulates that AhR mRNA levels could be used as an indicator of the exposure of clams' haemocytes to a mixture of xenobiotics such as chlorothalonil, mancozeb and BaP. However, further studies have to be pursued in order to unravel the molecular mechanisms involved in the p53 signaling pathway.