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.

Prevalence and determinants of SARS-CoV-2 neutralizing antibodies in Lebanon.

According to the Lebanese Ministry of Public Health, more than 1,053,000 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been confirmed in Lebanon so far. The actual number of cases is likely to be higher. We conducted a serological study from October 2020 to April 2021 to estimate the prevalence of SARS-CoV-2 neutralizing antibodies and identify associated factors. Serum samples as well as demographic, health, and behavioral data were collected from 2,783 subjects. Sera were tested by microneutralization assay. Neutralizing antibodies were detected in 58.9% of the study population. The positivity rate increased over the study period. It was highest among the group who remained at work during the COVID-19 pandemic and in peri-urban areas with limited adherence to preventive measures. Sex and age were associated with positivity. Reported previous COVID-19, exposure to a COVID-19 patient in the family, and attending gatherings were associated with increased prevalence. Not taking any precautionary measures against COVID-19 was a risk factor, whereas precautionary measures such as working from home and washing hands were protective. The high neutralizing antibody seroprevalence rates detected in this study emphasize the high transmission rate of SARS-CoV-2 infection in the community. Adherence to preventive measures and non-pharmaceutical interventions imposed by the government is recommended.

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.

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.

Immunogenicity and effectiveness of a bivalent influenza A/H1N2 vaccine strain against seasonal human influenza A viruses in mice.

BACKGROUND: Recent studies and reports have documented the ability of the co-circulating seasonal influenza A/H1N1 (ancestor: 2009 pandemic H1N1) and A/H3N2 to exchange their genetic segments, generating a novel H1N2 strain in different geographical localities around the world with an ability to infect human. This raises concerns and triggers alarms to develop a multivalent vaccine that can protect against the documented H1- and H3-type human influenza A viruses (IAVs). RESULTS: Here, we generated a PR8-based vaccine strain that carries the HA gene segment from the contemporary H1N1 virus while the NA gene segment was derived from a currently circulating influenza A/H3N2 strain. A recombinant PR8-based H1N2 vaccine strain (rgH1N2), engineered by reassortment between influenza A/H1N1 and A/H3N2 to mimic the documented human influenza A/H1N2, was used for immunization to provoke immunogenicity and cross-antigenicity against the H1- and H3-type human IAVs and was evaluated for its immunogenicity and effectiveness in mice. Following challenge infection of rgH1N2-vaccinated mice with contemporary influenza A/H1N1 and A/H3N2, results revealed that rgH1N2-vaccinated mice showed less viral shedding, more survival, and less body weight loss compared to control unvaccinated groups and vaccinated mice with rgH1N1 and rgH3N2. CONCLUSIONS: This study highlights the applicability of the PR8-based H1N2 vaccine strain to protect against seasonal IAVs and emphasizes the role of both surface proteins, HA and NA, to stimulate protective and neutralizing antibodies against circulating influenza A/H1N1 and A/H3N2 strains.

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.

In Vitro and In Vivo Antiviral Studies of New Heteroannulated 1,2,3-Triazole Glycosides Targeting the Neuraminidase of Influenza A Viruses.

There is an urgent need to develop and synthesize new anti-influenza drugs with activity against different strains, resistance to mutations, and suitability for various populations. Herein, we tested in vitro and in vivo the antiviral activity of new 1,2,3-triazole glycosides incorporating benzimidazole, benzooxazole, or benzotriazole cores synthesized by using a click approach. The Cu-catalyzation strategy consisted of 1,3-dipolar cycloaddition of the azidoalkyl derivative of the respective heterocyclic and different glycosyl acetylenes with five or six carbon sugar moieties. The antiviral activity of the synthesized glycosides against wild-type and neuraminidase inhibitor resistant strains of the avian influenza H5N1 and human influenza H1N1 viruses was high in vitro and in mice. Structure-activity relationship studies showed that varying the glycosyl moiety in the synthesized glycosides enhanced antiviral activity. The compound (2R,3R,4S,5R)-2-((1-(Benzo[d]thiazol-2-ylmethyl)-1H-1,2,3-triazol-4-yl)methoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Compound 9c) had a 50% inhibitory concentration (IC50) = 2.280 µM and a ligand lipophilic efficiency (LLE) of 6.84. The compound (2R,3R,4S,5R)-2-((1-((1H-Benzo[d]imidazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate had IC50 = 2.75 µM and LLE = 7.3 after docking analysis with the H5N1 virus neuraminidase. Compound 9c achieved full protection from H1N1 infection and 80% protection from H5N1 in addition to a high binding energy with neuraminidase and was safe in vitro and in vivo. This compound is suitable for further clinical studies as a new neuraminidase inhibitor.

Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b in Wild Birds and Live Bird Markets, Egypt.

Clade 2.3.4.4 H5Nx influenza viruses have further diversified into several subclades. Sub-clade 2.3.4.4b H5N1 viruses have been widely circulating in wild birds and detected in Europe, Africa, Asia, and North America since October 2020. In this study, we report the first detection of highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses in wild birds and domestic ducks from live bird markets in Egypt. Phylogenetic analysis revealed that the Egyptian H5N1 virus retained the genomic composition of Eurasian strains. Mutations in the viral proteins associated with zoonotic potential and pathogenicity were detected in Egyptian isolates. Egypt is considered a hot spot for the evolution of the influenza virus, so active surveillance of avian influenza viruses in Egypt is warranted.

SARS-CoV-2 Variants in Lebanon: Evolution and Current Situation.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seen a worldwide spread since its emergence in 2019, including to Lebanon, where 534,968 confirmed cases (8% of the population) and 7569 deaths have been reported as of 14 May 2021. With the genome sequencing of strains from various countries, several classification systems were established via genome comparison. For instance, the GISAID clades classification highlights key mutations in the encoded proteins that could potentially affect the virus' infectivity and transmission rates. In this study, 58 genomes of Lebanese SARS-CoV-2 strains were analyzed, 28 of which were sequenced for this study, and 30 retrieved from the GISAID and GenBank databases. We aimed to classify these strains, establish their phylogenetic relationships, and extract the mutations causing amino acid substitutions within, particularly, the structural proteins. The sequenced Lebanese SARS-COV-2 strains were classified into four GISAID clades and 11 Pango lineages. Moreover, 21 uncommon mutations in the structural proteins were found in the newly sequenced strains, underlining interesting combinations of mutations in the spike proteins. Hence, this study constitutes an observation and description of the current SARS-CoV-2 genetic and clade situation in Lebanon according to the available sequenced strains.

Immunogenicity and Safety of an Inactivated SARS-CoV-2 Vaccine: Preclinical Studies.

Since the emergence of SARS-CoV-2 at the end of 2019, 64 candidate vaccines are in clinical development and 173 are in the pre-clinical phase. Five types of vaccines are currently approved for emergency use in many countries (Inactivated, Sinopharm; Viral-vector, Astrazeneca, and Gamaleya Research Institute; mRNA, Moderna, and BioNTech/Pfizer). The main challenge in this pandemic was the availability to produce an effective vaccine to be distributed to the world's population in a short time. Herein, we developed a whole virus NRC-VACC-01 inactivated candidate SARS-CoV-2 vaccine and tested its safety and immunogenicity in laboratory animals. In the preclinical studies, we used four experimental animals (mice, rats, guinea pigs, and hamsters). Antibodies were detected as of week three post vaccination and continued up to week ten in the four experimental models. Safety evaluation of NRC-VACC-01 inactivated candidate vaccine in rats revealed that the vaccine was highly tolerable. By studying the effect of booster dose in the immunological profile of vaccinated mice, we observed an increase in neutralizing antibody titers after the booster shot, thus a booster dose was highly recommended after week three or four. Challenge infection of hamsters showed that the vaccinated group had lower morbidity and shedding than the control group. A phase I clinical trial will be performed to assess safety in human subjects.

FDA-Approved Drugs with Potent In Vitro Antiviral Activity against Severe Acute Respiratory Syndrome Coronavirus 2.

(1) Background: Drug repositioning is an unconventional drug discovery approach to explore new therapeutic benefits of existing drugs. Currently, it emerges as a rapid avenue to alleviate the COVID-19 pandemic disease. (2) Methods: Herein, we tested the antiviral activity of anti-microbial and anti-inflammatory Food and Drug Administration (FDA)-approved drugs, commonly prescribed to relieve respiratory symptoms, against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the viral causative agent of the COVID-19 pandemic. (3) Results: Of these FDA-approved antimicrobial drugs, Azithromycin, Niclosamide, and Nitazoxanide showed a promising ability to hinder the replication of a SARS-CoV-2 isolate, with IC50 of 0.32, 0.16, and 1.29 µM, respectively. We provided evidence that several antihistamine and anti-inflammatory drugs could partially reduce SARS-CoV-2 replication in vitro. Furthermore, this study showed that Azithromycin can selectively impair SARS-CoV-2 replication, but not the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). A virtual screening study illustrated that Azithromycin, Niclosamide, and Nitazoxanide bind to the main protease of SARS-CoV-2 (Protein data bank (PDB) ID: 6lu7) in binding mode similar to the reported co-crystalized ligand. Also, Niclosamide displayed hydrogen bond (HB) interaction with the key peptide moiety GLN: 493A of the spike glycoprotein active site. (4) Conclusions: The results suggest that Piroxicam should be prescribed in combination with Azithromycin for COVID-19 patients.

Common childhood vaccines do not elicit a cross-reactive antibody response against SARS-CoV-2.

Anecdotal evidence showed a negative correlation between Bacille Calmette-Guérin (BCG) vaccination and incidence of COVID-19. Incidence of the disease in children is much lower than in adults. It is hypothesized that BCG and other childhood vaccinations may provide some protection against SARS-CoV-2 infection through trained or adaptive immune responses. Here, we tested whether BCG, Pneumococcal, Rotavirus, Diphtheria, Tetanus, Pertussis, Hepatitis B, Haemophilus influenzae, Hepatitis B, Meningococcal, Measles, Mumps, and Rubella vaccines provide cross-reactive neutralizing antibodies against SARS-CoV-2 in BALB/c mice. Results indicated that none of these vaccines provided antibodies capable of neutralizing SARS-CoV-2 up to seven weeks post vaccination. We conclude that if such vaccines have any role in COVID-19 immunity, this role is not antibody-mediated.

Coding-Complete Genome Sequences of Two SARS-CoV-2 Isolates from Egypt.

This report announces the complete genome sequences of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolates detected in Egypt. The isolates were obtained from oropharyngeal swab specimens from two Egyptians in Upper and Lower Egypt. Sequence analysis showed mutations that differentiate Egyptian strains from the reference strain 2019-nCoV WHU01.

Prevalence of Severe Acute Respiratory Syndrome Coronavirus 2 Neutralizing Antibodies in Egyptian Convalescent Plasma Donors.

Using convalescent plasma as immunotherapy is an old method for treatment of infectious diseases. Several countries have recently allowed the use of such therapy for the treatment of COVID-19 patients especially those who are critically ill. A similar program is currently being tested in Egypt. Here, we tested 227 plasma samples from convalescent donors in Egypt for neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using a microneutralization (MN) assay. A third of the tested samples did not have antibody titers and 58% had titers between 1:10 and 1:80. Only 12% had titers >1:160. We also compared MN assays using different virus concentrations, plaque reduction neutralization (PRNT) assays, and a chemiluminescence assay that measures immunoglobulin G (IgG) binding to N and S proteins of SARS-CoV-2. Our results indicated that a MN assay using 100 TCID50/ml provides comparable results to PRNT and allows for high throughput testing.

Comparative Virological and Pathogenic Characteristics of Avian Influenza H5N8 Viruses Detected in Wild Birds and Domestic Poultry in Egypt during the Winter of 2016/2017.

The surveillance and virological characterization of H5N8 avian influenza viruses are important in order to assess their zoonotic potential. The genetic analyses of the Egyptian H5N8 viruses isolated through active surveillance in wild birds and domestic poultry in the winter of 2016/2017 showed multiple introductions of reassortant viruses. In this study, we investigated and compared the growth kinetics, infectivity, and pathogenicity of the three reassortant forms of H5N8 viruses detected in wild birds and domestic poultry in Egypt during the first introduction wave in the winter of 2016/2017. Three representative H5N8 viruses (abbreviated as 813, 871, and 13666) were selected. The 871/H5N8 virus showed enhanced growth properties in vitro in Madin Darby canine kidney (MDCK) and A549 cells. Interestingly, all viruses replicated well in mice without prior adaptation. Infected C57BL/6 mice showed 20% mortality for 813/H5N8 and 60% mortality for 871/H5N8 and 13666/H5N8, which could be attributed to the genetic differences among the viruses. Studies on the pathogenicity in experimentally infected ducks revealed a range of pathogenic effects, with mortality rate ranging from 0% for 813/H5N8 and 13666/H5N8 to 28% for 871/H5N8. No significant differences were observed among the three compared viruses in infected chickens. Overall, different H5N8 viruses had variable biological characteristics, indicating a continuous need for surveillance and virus characterization efforts.