Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Genetic and virological characteristics of a reassortant avian influenza A H6N1 virus isolated from wild birds at a live-bird market in Egypt.

The first detection of a human infection with avian influenza A/H6N1 virus in Taiwan in 2013 has raised concerns about this virus. During our routine surveillance of avian influenza viruses (AIVs) in live-bird markets in Egypt, an H6N1 virus was isolated from a garganey duck and was characterized. Phylogenetic analysis indicated that the Egyptian H6N1 strain A/Garganey/Egypt/20869C/2022(H6N1) has a unique genomic constellation, with gene segments inherited from different subtypes (H5N1, H3N8, H7N3, H6N1, and H10N1) that have been detected previously in AIVs from Egypt and some Eurasian countries. We examined the replication of kinetics of this virus in different mammalian cell lines (A549, MDCK, and Vero cells) and compared its pathogenicity to that of the ancestral H6N1 virus A/Quail/HK/421/2002(H6N1). The Egyptian H6N1 virus replicated efficiently in C57BL/6 mice without prior adaptation and grew faster and reached higher titers than in A549 cells than the ancestral strain. These results show that reassortant H6 AIVs might pose a potential threat to human health and highlight the need to continue surveillance of H6 AIVs circulating in nature.

Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Role of research Laboratories in pandemic and epidemic response in the Eastern Mediterranean Region: Experiences from COVID-19, avian influenza, and MERS-CoV.

We share the experience of research laboratories in the Eastern Mediterranean Region (EMR) that contributed to preparedness and response to highly pathogenic avian influenza (HPAI), Middle-East respiratory syndrome coronavirus (MERS-CoV), and coronavirus disease (COVID-19). Research groups in the region were pivotal in identifying, characterizing the pathogens and describing their evolution, distribution, transmission routes, and the immunological profile of exposed populations. They demonstrated the capacity to develop and test antivirals and potential vaccines. The EMR experience is a model of how national systems can work with researchers to improve regional preparedness and response to future epidemics and pandemics.

Potent Antiviral Activity of Vitamin B12 against Severe Acute Respiratory Syndrome Coronavirus 2, Middle East Respiratory Syndrome Coronavirus, and Human Coronavirus 229E.

Repurposing vitamins as antiviral supporting agents is a rapid approach used to control emerging viral infections. Although there is considerable evidence supporting the use of vitamin supplementation in viral infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the specific role of each vitamin in defending against coronaviruses remains unclear. Antiviral activities of available vitamins on the infectivity and replication of human coronaviruses, namely, SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), and human coronavirus 229E (HCoV-229E), were investigated using in silico and in vitro studies. We identified potential broad-spectrum inhibitor effects of Hydroxocobalamin and Methylcobalamin against the three tested CoVs. Cyanocobalamin could selectively affect SARS-CoV-2 but not MERS-CoV and HCoV-229E. Methylcobalamin showed significantly higher inhibition values on SARS-CoV-2 compared with Hydroxocobalamin and Cyanocobalamin, while Hydroxocobalamin showed the highest potent antiviral activity against MERS-CoV and Cyanocobalamin against HCoV-229E. Furthermore, in silico studies were performed for these promising vitamins to investigate their interaction with SARS-CoV-2, MERS-CoV, and HCoV-229E viral-specific cell receptors (ACE2, DPP4, and hAPN protein, respectively) and viral proteins (S-RBD, 3CL pro, RdRp), suggesting that Hydroxocobalamin, Methylcobalamin, and Cyanocobalamin may have significant binding affinity to these proteins. These results show that Methylcobalamin may have potential benefits for coronavirus-infected patients.

The episodic resurgence of highly pathogenic avian influenza H5 virus.

Highly pathogenic avian influenza (HPAI) H5N1 activity has intensified globally since 2021, increasingly causing mass mortality in wild birds and poultry and incidental infections in mammals1-3. However, the ecological and virological properties that underscore future mitigation strategies still remain unclear. Using epidemiological, spatial and genomic approaches, we demonstrate changes in the origins of resurgent HPAI H5 and reveal significant shifts in virus ecology and evolution. Outbreak data show key resurgent events in 2016-2017 and 2020-2021, contributing to the emergence and panzootic spread of H5N1 in 2021-2022. Genomic analysis reveals that the 2016-2017 epizootics originated in Asia, where HPAI H5 reservoirs are endemic. In 2020-2021, 2.3.4.4b H5N8 viruses emerged in African poultry, featuring mutations altering HA structure and receptor binding. In 2021-2022, a new H5N1 virus evolved through reassortment in wild birds in Europe, undergoing further reassortment with low-pathogenic avian influenza in wild and domestic birds during global dissemination. These results highlight a shift in the HPAI H5 epicentre beyond Asia and indicate that increasing persistence of HPAI H5 in wild birds is facilitating geographic and host range expansion, accelerating dispersion velocity and increasing reassortment potential. As earlier outbreaks of H5N1 and H5N8 were caused by more stable genomic constellations, these recent changes reflect adaptation across the domestic-bird-wild-bird interface. Elimination strategies in domestic birds therefore remain a high priority to limit future epizootics.

Comparison of SARS-Cov-2 omicron variant with the previously identified SARS-Cov-2 variants in Egypt, 2020-2022: insight into SARS-Cov-2 genome evolution and its impact on epidemiology, clinical picture, disease severity, and mortality.

BACKGROUND: The o severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic has killed millions of people and caused widespread concern around the world. Multiple genetic variants of SARS-CoV-2 have been identified as the pandemic continues. Concerns have been raised about high transmissibility and lower vaccine efficacy against omicron. There is an urgent need to better describe how omicron will impact clinical presentation and vaccine efficacy. This study aims at comparing the epidemiologic, clinical, and genomic characteristics of the omicron variant prevalent during the fifth wave with those of other VOCs between May 2020 and April 2022. METHODS: Epidemiological data were obtained from the National Electronic Diseases Surveillance System. Secondary data analysis was performed on all confirmed COVID-19 patients. Descriptive data analysis was performed for demographics and patient outcome and the incidence of COVID-19 was calculated as the proportion of SARS-CoV-2 confirmed patients out of the total population of Egypt. Incidence and characteristics of the omicron cohort from January- April 2022, were compared to those confirmed from May 2020-December 2021. We performed the whole-genome sequencing of SARS-CoV-2 on 1590 specimens using Illumina sequencing to describe the circulation of the virus lineages in Egypt. RESULTS: A total of 502,629 patients enrolled, including 60,665 (12.1%) reported in the fifth wave. The incidence rate of omicron was significantly lower than the mean of incidences in the previous subperiod (60.1 vs. 86.3/100,000 population, p < 0.001). Symptoms were reported less often in the omicron cohort than in patients with other variants, with omicron having a lower hospitalization rate and overall case fatality rate as well. The omicron cohort tended to stay fewer days at the hospital than did those with other variants. We analyzed sequences of 2433 (1590 in this study and 843 were obtained from GISAID platform) Egyptian SARS-CoV-2 full genomes. The first wave that occurred before the emergence of global variants of concern belonged to the B.1 clade. The second and third waves were associated with C.36. Waves 4 and 5 included B.1.617.2 and BA.1 clades, respectively. CONCLUSIONS: The study indicated that Omicron-infected patients had milder symptoms and were less likely to be hospitalized; however, patients hospitalized with omicron had a more severe course and higher fatality rates than those hospitalized with other variants. Our findings demonstrate the importance of combining epidemiological data and genomic analysis to generate actionable information for public health decision-making.

Detection of Coronaviruses in Bats in Lebanon during 2020.

Bats are considered the main reservoir of coronaviruses (CoVs), and research evidence suggests the essential role of bats in the emergence of Severe Acute Respiratory Syndrome Coronaviruses (SARS-CoV) and SARS-CoV-2. SARS-CoV-like viruses have been recently detected in bats in different countries. In 2020, we conducted surveillance for CoVs among six different bat species in Lebanon. Of 622 swab specimens taken, 77 tested positive. Alpha- and Beta- CoVs were identified in samples collected from different species. Our results show that SARS-like coronaviruses circulate in bats in this region, and we provide new data on their genetic diversity. The interaction between the spike of the detected SARS-CoV-like viruses and the human angiotensin-converting enzyme 2 (hACE2) receptor could be crucial in understanding the origin of the epidemic. The 3D protein structure analysis revealed that the receptor-binding domains of the SARS-like virus identified in Lebanon bind to the hACE2 protein more efficiently than to the spike of the SARS-CoV-2 strain. The spike of the detected SARS-CoV-like viruses does not contain the recognition site of furin at the cleavage site. Thus, our study highlights the variety of bat coronaviruses in Lebanon and suggests the zoonotic potential for other SARS-CoV-like viruses.

Field Research Is Essential to Counter Virological Threats.

The interface between humans and wildlife is changing and, with it, the potential for pathogen introduction into humans has increased. Avian influenza is a prominent example, with an ongoing outbreak showing the unprecedented expansion of both geographic and host ranges. Research in the field is essential to understand this and other zoonotic threats. Only by monitoring dynamic viral populations and defining their biology in situ can we gather the information needed to ensure effective pandemic preparation.

State and situation of avian influenza in the Eastern Mediterranean Region.

Avian influenza viruses have had a significant burden of disease on animal and public health in countries of the Eastern Mediterranean Region. In this review, we aimed at describing the state of avian influenza in the region from 2011 to 2021. We gathered information available through the peer-reviewed scientific literature, public gene sequence depositories, OIE World Animal Health Information System platform, World Health Organization FluNet, Joint External Evaluation reports, and governmental, Food and Agriculture Organization of the United Nations, and World Organization for Animal Health websites. We used an interdisciplinary perspective consistent with the One Health approach to perform a qualitative synthesis and making recommendations. Analysis showed that although avian influenza research in the Eastern Mediterranean Region has gained more attention during the last decade, it was limited to only few countries and to basic science research. Data highlighted the weakness in surveillance systems and reporting platforms causing underestimation of the actual burden of disease among humans and animals. Inter-sectoral communication and collaboration for avian influenza prevention, detection, and response remain weak. Influenza surveillance at the human-animal interface and the application of the One Health paradigm are lacking. Countries' animal health and public health sectors rarely publish their surveillance data and findings. This review suggested that surveillance at the human-animal interface, research, and reporting capacities should be enhanced to improve understanding and control of avian influenza in the region. Implementing a rapid and comprehensive One Health approach for zoonotic influenza in the Eastern Mediterranean Region is recommended.

The species coalescent indicates possible bat and pangolin origins of the COVID-19 pandemic.

A consensus species tree is reconstructed from 11 gene trees for human, bat, and pangolin beta coronaviruses from samples taken early in the pandemic (prior to April 1, 2020). Using coalescent theory, the shallow (short branches relative to the hosts) consensus species tree provides evidence of recent gene flow events between bat and pangolin beta coronaviruses predating the zoonotic transfer to humans. The consensus species tree was also used to reconstruct the ancestral sequence of human SARS-CoV-2, which was 2 nucleotides different from the Wuhan sequence. The time to most recent common ancestor was estimated to be Dec 8, 2019 with a bat origin. Some human, bat, and pangolin coronavirus lineages found in China are phylogenetically distinct, a rare example of a class II phylogeography pattern (Avise et al. in Ann Rev Eco Syst 18:489-422, 1987). The consensus species tree is a product of evolutionary factors, providing evidence of repeated zoonotic transfers between bat and pangolin as a reservoir for future zoonotic transfers to humans.

Comparative pathogenic potential of avian influenza H7N3 viruses isolated from wild birds in Egypt and their sensitivity to commercial antiviral drugs.

Active surveillance and studying the virological features of avian-origin influenza viruses are essential for early warning and preparedness for the next potential pandemic. During our active surveillance of avian influenza viruses in wild birds in Egypt in the period 2014-2017, multiple reassortant low-pathogenic avian influenza H7N3 viruses were isolated. In this study, we investigated and compared the infectivity, pathogenicity, and transmission of four different constellation forms of Egyptian H7N3 viruses in chickens and mice and assessed the sensitivity of these viruses to different commercial antiviral drugs in vitro. Considerable variation in virus pathogenicity was observed in mice infected with different H7N3 viruses. The mortality rate ranged from 20 to 100% in infected mice. Infected chickens showed only ocular clinical signs at three days postinfection as well as systemic viral infection in different organs. Efficient virus replication and transmission in chickens was observed within each group, indicating that these subtypes can spread easily from wild birds to poultry without prior adaptation. Mutations in the viral proteins associated with antiviral drug resistance were not detected, and all strains were sensitive to the antiviral drugs tested. In conclusion, all of the viruses studied had the ability to infect mice and chickens. H7N3 viruses circulating among wild birds in Egypt could threaten poultry production and public health.

Molecular identification and virological characteristics of highly pathogenic avian influenza A/H5N5 virus in wild birds in Egypt.

Multiple incursions of different subtypes of highly pathogenic avian influenza (HPAI) A/H5NX viruses have caused widely considerable outbreaks in poultry and hundreds of human infections. Extensive reassortment events associated with currently circulating clade 2.3.4.4b of A/H5NX viruses have been widely recorded. Wild migratory birds contribute to the spillover of diverse viruses throughout their migration flyways. During our active surveillance of avian influenza in Egypt, we successfully isolated and fully characterized HPAI A/H5N5 virus of clade 2.3.4.4b that was detected in a healthy purple heron. The Egyptian H5N5 virus is genotypically similar with the same subtype that was detected in the far east of Russia and several European countries. The antigenic analysis showed that the Egyptian H5N5 virus is distinct from HPAI A(H5N8) viruses in Egypt. The virus preferentially binds to avian-like receptors rather than human-like receptors. Our results showed that the virus caused 100% and 60% lethality in chicken and mice respectively. Increasing active surveillance efforts, monitoring the dynamics of emerging AIVs, and risk assessment implementation should be globally applied especially in hot spot regions like Egypt.

Designing and validating a One Health Research Translation Framework through literature-based case studies in Egypt.

Within One Health, research translation is a dynamic process involving collaboration and communication between the human, animal, and environmental health sectors to create and apply research findings to address health threats at the human-animal-environment interface. Research translation is essential for the creation of evidence-based policies and programs for the prevention and control of infectious diseases and other health threats, and thus is an important component of a robust national capacity to effectively prevent, control, and mitigate biological incidents. However, there is a lack of conceptual guidance and training materials for research translation in a One Health context. To address this need, we developed a novel One Health Research Translation Framework that describes an iterative process for research and policy stakeholders to collaborate to design and implement research applications addressing One Health zoonotic disease challenges. In addition, we developed accompanying training materials to validate the Framework and facilitate capacity building for understanding and applying research translation concepts to zoonotic disease threats. The training materials consist of exercises to map One Health communication pathways and literature-based case studies on research translation to address zoonotic disease concerns. The Framework and training materials were piloted with Egyptian One Health stakeholders at a workshop in Cairo in 2018. The outcomes of the workshop validated the comprehensiveness and applicability of the Framework and training materials, as participants were able to demonstrate a firm understanding of research translation processes and successfully apply research translation and One Health concepts to real-world zoonotic disease scenarios. Overall, the Framework and accompanying training materials address an important gap in capacity building for One Health stakeholders and are valuable tools for strengthening research translation networks that promote development of innovative, evidence-based solutions to One Health zoonotic disease threats.

Insights into Genetic Characteristics and Virological Features of Endemic Avian Influenza A (H9N2) Viruses in Egypt from 2017-2021.

From 2010 to 2013, genotype I avian influenza A(H9N2) viruses of the G1-lineage were isolated from several poultry species in Egypt. In 2014, novel reassortant H9N2 viruses were detected in pigeons designated as genotype II. To monitor the subsequent genetic evolution of Egyptian A(H9N2) viruses, we characterized the full genomes of 173 viruses isolated through active surveillance from 2017 to 2022. In addition, we compared the virological characteristics and pathogenicity of representative viruses. Phylogenetic analysis of the HA indicated that all studied sequences from 2017-2021 were grouped into G1-like H9N2 viruses previously detected in Egypt. Phylogenetic analysis indicated that the Egyptian A(H9N2) viruses had undergone further reassortment, inheriting four genes (PB2, PB1, PA, NS) from genotype II, with their remaining segments deriving from genotype I viruses (these viruses designated as genotype III). Studying the virological features of the two most dominant genotypes (I and III) of Egyptian H9N2 viruses in vitro and in vivo indicated that both replicated well in mammalian cells, but did not show any clinical signs in chickens, ducks, and mice. Monitoring avian influenza viruses through surveillance programs and understanding the genetic and antigenic characteristics of circulating H9N2 viruses are essential for risk assessment and influenza pandemic preparedness.

Insight into Genetic Characteristics of Identified SARS-CoV-2 Variants in Egypt from March 2020 to May 2021.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was first detected in Egypt in February 2020. Data about the prevalence rates of the SARS-CoV-2 lineages are relatively scarce. To understand the genetic characteristics of SARS-CoV-2 in Egypt during several waves of the pandemic, we analyzed sequences of 1256 Egyptian SARS-CoV-2 full genomes from March 2020 to May 2021. From one wave to the next, dominant strains have been observed to be replaced by other dominant strains. We detected an emerging lineage of SARS-CoV-2 in Egypt that shares mutations with the variant of concern (VOC). The neutralizing capacity of sera collected from cases infected with C.36.3 against dominant strains detected in Egypt showed a higher cross reactivity of sera with C.36.3 compared to other strains. Using in silico tools, mutations in the spike of SARS-CoV-2 induced a difference in binding affinity to the viral receptor. The C.36 lineage is the most dominant SARS-CoV-2 lineage in Egypt, and the heterotrophic antigenicity of SARS-CoV-2 variants is asymmetric. These results highlight the value of genetic and antigenic analyses of circulating strains in regions where published sequences are limited.

Robust antiviral activity of commonly prescribed antidepressants against emerging coronaviruses: in vitro and in silico drug repurposing studies.

During the current coronavirus disease 2019 (COVID-19) pandemic, symptoms of depression are commonly documented among both symptomatic and asymptomatic quarantined COVID-19 patients. Despite that many of the FDA-approved drugs have been showed anti-SARS-CoV-2 activity in vitro and remarkable efficacy against COVID-19 in clinical trials, no pharmaceutical products have yet been declared to be fully effective for treating COVID-19. Antidepressants comprise five major drug classes for the treatment of depression, neuralgia, migraine prophylaxis, and eating disorders which are frequently reported symptoms in COVID-19 patients. Herein, the efficacy of eight frequently prescribed FDA-approved antidepressants on the inhibition of both SARS-CoV-2 and MERS-CoV was assessed. Additionally, the in vitro anti-SARS-CoV-2 and anti-MERS-CoV activities were evaluated. Furthermore, molecular docking studies have been performed for these drugs against the spike (S) and main protease (Mpro) pockets of both SARS-CoV-2 and MERS-CoV. Results showed that Amitriptyline, Imipramine, Paroxetine, and Sertraline had potential anti-viral activities. Our findings suggested that the aforementioned drugs deserve more in vitro and in vivo studies targeting COVID-19 especially for those patients suffering from depression.

Induced humoral immunity of different types of vaccines against most common variants of SARS-CoV-2 in Egypt prior to Omicron outbreak.

The diversity of SARS-CoV-2 continues to lead to the emergence of new SARS-CoV-2 variants. SARS-CoV-2 antibody assays are crucial in managing the COVID-19 pandemic by determining the neutralizing antibody response. This study aims to investigate vaccine-induced antibodies against most common variants of SARS-CoV-2 in Egypt. Sera samples were collected from vaccinated participants and neutralizing activity against the SARS-CoV-2 variants was determined using microneutralization assay. Our results show that the BNT162b2 (Pfizer-BioNTech), ChAdOx1 nCov-19 (AstraZeneca), and Ad26.COV2.S COVID-19 (Janssen) vaccines elicited neutralizing antibody responses more than the BBIBP-CorV vaccine (Sinopharm) against B.1, C.36.3, and AY.32 (Delta) variants. While vaccines remain highly effective in managing the COVID-19 pandemic, ongoing monitoring of vaccine effectiveness is needed.