Rohaim, M. A., M. Q. Al-Natour, M. A. Abdelsabour, R. F El Naggar, Y. M. Madbouly, K. A. Ahmed, and M. Munir, "Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses.", International journal of molecular sciences, vol. 22, issue 16, 2021. Abstract

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 10 egg infective dose 50 (EID) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID 10), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.

Rohaim, M. A., R. E. F. Naggar, M. A. Abdelsabour, B. A. Ahmed, M. M. Hamoud, K. A. Ahmed, O. K. Zahran, and M. Munir, "Insights into the Genetic Evolution of Duck Hepatitis A Virus in Egypt.", Animals : an open access journal from MDPI, vol. 11, issue 9, 2021. Abstract

Duck hepatitis virus (DHV) is one of the commercially important diseases of ducklings worldwide. It is an acute and highly infectious disease of ducklings caused by three different serotypes (1-3) of duck hepatitis A virus (DHAV), and serotype 1 is the most common in poultry. To date, little is known about the prevalence and genetic characterisation of DHAV-1 in Egypt. In the current study, isolation and complete genomic analyses of DHAVs circulating in commercial duck farms in different Egyptian governorates were conducted. A total of eighteen samples were collected from six Egyptian governorates of 3-11 days old ducklings (Pekin and Mullard) with a history of nervous signs and high mortality rates. Five out of eighteen (5/18) samples were screened positive for the DHAV-1 based on the VP1 gene. These samples were individually used for virus isolation in embryonated duck embryos (EDE), followed by complete genome sequencing. Phylogenomic analyses showed that DHAV serotype I; genotype I were diversified into four different groups (1-4). Most of the recent circulating Egyptian DHAV strains are clustered within group 4, while isolates characterised within this study were clustered within group 1. Recombination analyses revealed that the emergence of a new recombinant virus-DHAV-1 strain Egypt-10/2019-through recombination. Likewise, the selective pressure analyses showed the existence, inside or near areas of the viral attachment or related functions, of positive scores highlighting the importance of natural selection and viral evolution mechanism at different protein domains. The findings of this study provide updated information on the epidemiological and genetic features of DHAV-1 strains and underscore the importance of DHAV surveillance as well as re-evaluation for currently used vaccines.

Park, J. - G., F. S. Oladunni, M. A. Rohaim, J. Whittingham-Dowd, J. Tollitt, M. D. J. Hodges, N. Fathallah, M. B. Assas, W. Alhazmi, A. Almilaibary, et al., "Immunogenicity and protective efficacy of an intranasal live-attenuated vaccine against SARS-CoV-2.", iScience, vol. 24, issue 9, pp. 102941, 2021. Abstract

Global deployment of an effective and safe vaccine is necessary to curtail the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a Newcastle disease virus (NDV)-based vectored-vaccine in mice and hamsters for its immunogenicity, safety, and protective efficacy against SARS-CoV-2. Intranasal administration of recombinant (r)NDV-S vaccine expressing spike (S) protein of SARS-CoV-2 to mice induced high levels of SARS-CoV-2-specific neutralizing immunoglobulin A (IgA) and IgG2a antibodies and T-cell-mediated immunity. Hamsters immunized with two doses of vaccine showed complete protection from lung infection, inflammation, and pathological lesions following SARS-CoV-2 challenge. Importantly, administration of two doses of intranasal rNDV-S vaccine significantly reduced the SARS-CoV-2 shedding in nasal turbinate and lungs in hamsters. Collectively, intranasal vaccination has the potential to control infection at the site of inoculation, which should prevent both clinical disease and virus transmission to halt the spread of the COVID-19 pandemic.

Rohaim, M. A., R. F El Naggar, Y. Madbouly, M. A. Abdelsabour, K. A. Ahmed, and M. Munir, "Comparative infectivity and transmissibility studies of wild-bird and chicken-origin highly pathogenic avian influenza viruses H5N8 in chickens.", Comparative immunology, microbiology and infectious diseases, vol. 74, pp. 101594, 2021. Abstract

Despite the recent advances in avian influenza viruses surveillance and genomic data, fundamental questions concerning the ecology and evolution of these viruses remain elusive. In Egypt, H5N8 highly pathogenic avian influenza viruses (HPAIVs) are co-circulating simultaneously with HPAIVs of subtypes H5N1 and low-pathogenic avian influenza viruses (LPAIVs) of subtype H9N2 in both commercial and backyard poultry. In order to isolate AIVs from wild birds and to assess their potential in causing infection in commercial poultry, a total of thirty-four cloacal swab samples were collected from apparently healthy migratory wild birds (Anas acuta, Anas crecca, Rallus aquaticus, and Bubulcus ibis) from four Egyptian Governorates (Giza, Menoufia, Gharbia, and Dakahlia). Based on matrix (M) gene-targeting real-time reverse transcriptase PCR and subsequent genetic characterization, our results revealed two positive isolates (2/34) for H5N8 whereas no H5N1 and H9N2 subtypes were detected. Genetic characterization of the full-length haemagglutinin (HA) genes revealed the clustering of two reported isolates within genotype 5 of clade 2.3.4.4b. The potential of a wild bird-origin H5N8 virus isolated from a cattle egret for its transmission capability within and between chickens was investigated in compare to chicken origin H5N8 AIV. Chickens inoculated with cattle egret isolate showed varying clinical signs and detection of virus shedding. In contrast, the contact chickens showed less levels of virus secretion indicating efficient virus inter/intra-species transmission. These results demonstrated the possibility for spreading of wild bird origin H5N8 viruses between chicken. In conclusion, our study highlights the need for continuous and frequent monitoring of the genetic diversity of H5N8 AIVs in wild birds as well as commercial poultry sectors for better understanding and determining the genetic nature of these viruses, which is fundamental to predict any future threat through virus reassortment with the potential to threaten human and animal health. Likewise, an assessment of coverage and efficacy of different vaccines and or vaccination regimes in the field conditions should be reconsidered along with strict biosecurity measures.

Abdel-Sabour, M. A., M. A. Rohaim, O. J. A. Salman, Samah E Abodalal, F. F. Mohammad, M. S. Madkour, N. A. Abdel-Wanis, and M. Munir, "Immunogenicity and efficacy of a bivalent vaccine against infectious bronchitis virus.", Comparative immunology, microbiology and infectious diseases, vol. 77, pp. 101670, 2021. Abstract

Infectious bronchitis (IB) is a highly contagious viral disease and is responsible for considerable economic losses in the poultry industry, worldwide. To mitigate the IB-associated losses, multiple vaccines are being applied in the sector with variable successes and thus necessitating the development of a potent vaccine to protect against the IB in the poultry. In the present study, we investigated a bivalent live attenuated vaccine consisting of IB virus (IBV) strain H120 (GI-1 lineage) and D274 (GI-12 lineage) to evaluate its protection against heterologous variant of IBV (GI-23 lineage) in chicken. Protection efficacy was evaluated based on the serology, clinical signs, survival rates, tracheal and kidney histopathology and the viral shedding. Results demonstrated that administering live H120 and D274 (named here Classivar®) vaccine in one day-old and 14 days-old provided 100 % protection. We observed a significant increase in the mean antibody titers, reduced virus shedding, and ameliorated histopathology lesions compared to routinely used vaccination regimes. These results revealed that usage of different IBV vaccines combination can successfully ameliorate the clinical outcome and pathology in vaccinated chicks especially after booster vaccination regime using Classivar®. In conclusions, our data indicate that Classivar® vaccine is safe in chicks and may serve as an effective vaccine against the threat posed by commonly circulating IBV strains in the poultry industry.

Xiao, S., S. Wang, D. Jiang, X. Cheng, X. Zhu, F. Lin, B. Yu, H. Dong, X. Wang, M. Munir, et al., "VP2 virus-like particles elicit protective immunity against duckling short beak and dwarfism syndrome in ducks.", Transboundary and emerging diseases, 2021. Abstract

Duckling short beak and dwarfism syndrome virus (SBDSV), an emerging goose parvovirus, has caused short beak and dwarfism syndrome (SBDS) in Chinese duck flocks since 2015. Presently, there is no commercial vaccine against SBDS. In the present study, a virus-like particle (VLP)-based candidate vaccine was developed against this disease. A baculovirus expression system was used to express the SBDSV VP2 protein in Sf9 cells. Immunofluorescence assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were used to confirm protein expression. Furthermore, transmission electron microscopy was used to observe the formation of VLPs. VLPs were formulated into an oil-adjuvanted maternal vaccine to evaluate humoral responses in breeding ducks via latex particle agglutination inhibition assay (LPAI) and microneutralization assay. The offspring were challenged with SBDSV to test the protective efficacy. A single dose of SBDSV was able to induce the high level of LPAI antibodies in ducks, with LPAI and neutralization peak titres of 4.9 ± 1.20 log2 and 7.1 ± 1.20 log2, respectively, at 4 weeks post-vaccination (wpv). The average LPAI titre of yolk antibodies in duck eggs receiving 2 doses (first and boost doses) of the vaccine was 5.3 ± 1.09 log2 at 4 weeks post-boost. The protective efficacy of the maternal vaccine was 87.5%-100%. These results indicate that SBDSV VLPs can be a promising vaccine candidate for controlling SBDS.

Naggar, R. E. F., M. A. Rohaim, and M. Munir, "Potential reverse spillover of infectious bursal disease virus at the interface of commercial poultry and wild birds.", Virus genes, vol. 56, issue 6, pp. 705-711, 2020. Abstract

Recently, multiple spillover events between domesticated poultry and wild birds have been reported for several avian viruses. This phenomenon highlights the importance of the livestock-wildlife interface in the possible emergence of novel viruses. The aim of the current study was to investigate the potential spillover and epidemiological links of infectious bursal disease virus (IBDV) between wild birds and domestic poultry. To this end, twenty-eight cloacal swabs were collected from four species of free-living Egyptian wild birds (i.e. mallard duck, bean goose, white-fronted goose and black-billed magpie). Genetic and phylogenetic analysis of three positive isolates revealed that the IBDV/USC-1/2019 strain clustered with previously reported very virulent IBDV (vvIBDV) Egyptian isolates. Interestingly, two other wild bird-origin isolates (i.e. IBDV/USC-2/2019 and IBDV/USC-3/2019) grouped with a vaccine strain that is being used in commercial poultry. In conclusion, our results revealed the molecular detection of vaccine and vvIBDV-like strains in Egyptian wild birds and highlighted the potential role of wild birds in IBDV epidemiology in disease-endemic regions.

Rohaim, M. A., R. F El Naggar, E. Clayton, and M. Munir, "Structural and functional insights into non-structural proteins of coronaviruses.", Microbial pathogenesis, vol. 150, pp. 104641, 2020. Abstract

Coronaviruses (CoVs) are causing a number of human and animal diseases because of their zoonotic nature such as Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). These viruses can infect respiratory, gastrointestinal, hepatic and central nervous systems of human, livestock, birds, bat, mouse, and many wild animals. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerging respiratory virus and is causing CoVID-19 with high morbidity and considerable mortality. All CoVs belong to the order Nidovirales, family Coronaviridae, are enveloped positive-sense RNA viruses, characterised by club-like spikes on their surfaces and large RNA genome with a distinctive replication strategy. Coronavirus have the largest RNA genomes (~26-32 kilobases) and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. Non-structural proteins (nsp) 7-16 are cleaved from two large replicase polyproteins and guide the replication and processing of coronavirus RNA. Coronavirus replicase has more or less universal activities, such as RNA polymerase (nsp 12) and helicase (nsp 13), as well as a variety of unusual or even special mRNA capping (nsp 14, nsp 16) and fidelity regulation (nsp 14) domains. Besides that, several smaller subunits (nsp 7- nsp 10) serve as essential cofactors for these enzymes and contribute to the emerging "nsp interactome." In spite of the significant progress in studying coronaviruses structural and functional properties, there is an urgent need to understand the coronaviruses evolutionary success that will be helpful to develop enhanced control strategies. Therefore, it is crucial to understand the structure, function, and interactions of coronaviruses RNA synthesizing machinery and their replication strategies.

Rohaim, M. A., R. F El Naggar, M. A. Abdelsabour, M. H. A. Mohamed, I. M. El-Sabagh, and M. Munir, "Evolutionary Analysis of Infectious Bronchitis Virus Reveals Marked Genetic Diversity and Recombination Events.", Genes, vol. 11, issue 6, pp. 605, 2020. Abstractgenes-11-00605-v2.pdf

In the last 5 years, frequent outbreaks of infectious bronchitis virus (IBV) are observed in both broiler and layer chicken flocks in the Kingdom of Saudi Arabia (KSA) in spite of extensive usage of vaccines. The IBV is a widespread avian coronavirus affecting both vaccinated and unvaccinated chicken flocks and is attributed to significant economic losses, around the globe. In the present study, 58 ( = 58) samples were collected from four different commercial poultry flocks from 8 KSA districts during 2019. A total of nine positive isolates (9/58; 15.5%), based on real-time reverse transcriptase PCR targeting nucleocapsid (N) gene, were used for further genetic characterization and evolutionary analysis. Genetic characterization of the partial spike (S1) gene revealed the clustering of the reported isolates into three different genotypes, whereas four additional isolates were grouped within 4/91 genotype, two isolates within IS/885 genotype, one isolate was closely related to IS/1494/06, and two isolates were grouped within classic serotype (vaccine-like strains). Phylodynamic revealed clustering of four isolated viruses within GI-13 lineage, three isolates within GI-23 lineage, and two isolates within GI-1 lineage. Results indicate that there are high evolutionary distances between the newly identified IBV strains in this study and the commercially used vaccines (GI-1), suggesting that IBV strains circulating in the KSA are under constant evolutionary pressures. Selective pressure biostatistics analyses consistently demonstrate the presence of a higher positive score which highlights the role of natural selection, a mechanism of virus evolution on sites located on the protein surface, within or nearby domains involved in viral attachment or related functions. Recombination analysis revealed emergence of two isolates through recombination events resulting in new recombinant viruses. Taken together, these finding demonstrate the genetic and evolutionary insights into the currently circulating IBV genotypes in KSA, which could help to better understand the origin, spread, and evolution of infectious bronchitis viruses, and to ascertain the importance of disease monitoring as well as re-evaluation for the currently used vaccines and vaccination programs.

Rohaim, M. A., R. F El Naggar, M. M. Hamoud, A. - H. I. Bazid, A. M. Gamal, S. E. Laban, M. A. Abdel-Sabour, S. A. E. Nasr, M. M. Zaki, M. Z. Shabbir, et al., "Emergence and genetic analysis of variant pathogenic 4/91 (serotype 793/B) infectious bronchitis virus in Egypt during 2019.", Virus genes, vol. 55, issue 5, pp. 720-725, 2019. Abstract

Infectious bronchitis virus (IBV) affects both vaccinated and unvaccinated flocks worldwide, with a significant impact on the poultry industry. The aim of the present study is to characterize an emerging variant pathogenic IBV originating from field outbreaks in vaccinated Egyptian layer flock. Samples were collected from disease-suspected flock with a history of administration of live and inactivated IBV vaccines (Ma5 type). Virus propagation in embryonated chicken eggs (ECEs), after three successive passages, revealed typical IBV lesions such as curling and dwarfism. The reported isolate was identified by a real-time reverse transcriptase PCR assay targeting nucleocapsid (N) gene and, further characterized by full-length spike (S1) gene sequencing. Phylogenetic analysis revealed clustering of the isolated virus within 4/91 genotype of GI-13 lineage. Deduced amino acid sequences identity revealed 75-76% and 88-90% similarity with the currently used classic (H120, Ma5, and M41) and variant vaccine strains (4/91 and CR88) in Egypt, respectively. Recombination analysis gave an evidence for distinct patterns of origin for the studied isolate providing another example of intra-genotypic recombination among IBVs and the first example of recombination within the GI-13 lineage in the Egyptian field. The studied isolate (IBV/CK/EG/Fadllah-10/2019) emerged as a result of recombination between the variant group (Egy/var I genotype, GI-23 lineage) as a major parent and the CR88 variant vaccine strain (4/91 genotype, GI-13 lineage) as minor parent. Our data suggest that both mutation and recombination may be contributing to the emergence of IBV variants which ascertain the importance of disease monitoring in vaccinated flocks as well as re-appropriation for the current vaccine strategies.