Structural and functional insights into non-structural proteins of coronaviruses

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. © 202

Comparative infectivity and transmissibility studies of wild-bird and chicken-origin highly pathogenic avian influenza viruses H5N8 in chickens

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

Transgenic chicks expressing interferon-inducible transmembrane protein 1 (Ifitm1) restrict highly pathogenic h5n1 influenza viruses

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 sarcomaleukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 104 egg infective dose 50 (EID50 ) 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 (EID50 105 ), 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. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Insights into the genetic evolution of duck hepatitis a virus in Egypt

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 sero-types (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 his-tory 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. Phylo-genomic analyses showed that DHAV serotype I; genotype I were diversified into four different groups (1, 2, 3 and 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

Potential reverse spillover of infectious bursal disease virus at the interface of commercial poultry and wild birds

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

Genome-wide classification of type I, type II and type III interferon-stimulated genes in chicken fibroblasts

Interferons (IFNs) play central roles in establishing innate immunity and mediating adaptive immunity against multiple pathogens. Three known types of IFNs identify their cognate receptors, initiate cascades of signalling events and eventually result in the induction of a myriad of IFN-stimulated genes (ISGs). These ISGs perform a multitude of functions and cumulatively corroborate a bespoke antiviral state to safeguard hosts against invading viruses. Owing to the unique nature of a chicken’s immune system and the lack of foundational profiling information on the nature and dynamic expression of IFN-specific ISGs at the genome scale, we performed a systematic and extensive analysis of type I, II and III IFN-induced genes in chicken. Employing pan-IFN responsive chicken fibroblasts coupled with transcriptomics, we observed an over-representation of up-regulated ISGs compared to down-regulated ISGs by all types of IFNs. Intriguingly, prediction of IFN-stimulated response element (ISRE) and gamma-IFN activation sequence (GAS) revealed a substantial number of GAS motifs in selective and significantly induced ISGs in chicken. Extensive comparative, genome-wide and differential expression analysis of ISGs under equivalent signalling input catalogue a set of genes that were either IFN-specific or independent of types of IFNs used to prime fibroblasts. These comprehensive datasets, first of their kinds in chicken, will establish foundations to elucidate the mechanisms of actions and breadth of antiviral action of ISGs, which may propose alternative avenues for targeted antiviral therapy against viruses of poultry of public health importance

Avian Orthoavulavirus Type-1 as Vaccine Vector against Respiratory Viral Pathogens in Animal and Human

Avian orthoavulaviruses type-1 (AOaV-1) have recently transitioned from animal vaccine vector to a bona fide vaccine delivery vehicle in human. Owing to induction of robust innate and adaptive immune responses in mucus membranes in both birds and mammals, AOaVs offer an at-tractive vaccine against respiratory pathogens. The unique features of AOaVs include over 50 years of safety profile, stable expression of foreign genes, high infectivity rates in avian and mammalian hosts, broad host spectrum, limited possibility of recombination and lack of pre-existing immunity in humans. Additionally, AOaVs vectors allow the production of economical and high quantities of vaccine antigen in chicken embryonated eggs and several GMP-grade mammalian cell lines. In this review, we describe the biology of AOaVs and define protocols to manipulate AOaVs genomes in effectively designing vaccine vectors. We highlighted the potential and established portfolio of AOaV-based vaccines for multiple respiratory and non-respiratory viruses of veterinary and medical importance. We comment on the limitations of AOaV-based vaccines and propose mitigations strategies. The exploitation of AOaVs vectors is expanding at an exciting pace; thus, we have limited the scope to their use as vaccines against viral pathogens in both animals and humans

The Molecular Virology of Coronaviruses with Special Reference to SARS-CoV-2

Introduction: Coronaviruses (CoVs) are large, enveloped and positive-sense RNA viruses which are responsible for a range of upper respiratory and digestive tract infections. Interest in coronaviruses has recently escalated due to the identification of a newly emerged coronavirus named severe acute respiratory syndrome 2 (SARS-CoV-2), which is the causative agent of the COVID-19 pandemic. In this chapter, we summarise molecular virological features of coronaviruses and understand their molecular mechanisms of replication in guiding the control of the global COVID-19 pandemic.Methods: We applied a holistic and comparative approach to assess the current understanding of coronavirus molecular virology and identify research gaps among different human coronaviruses.Results: Coronaviruses can utilise unique strategies that aid in their pathogenicity, replication and survival in multiple hosts. Replication of coronaviruses involves novel mechanisms such as ribosomal frameshifting and the synthesis of both genomic and sub-genomic RNAs. We summarised the key components in coronavirus molecular biology and molecular determinants of pathogenesis. Focusing largely on SARS-CoV-2 due to its current importance, this review explores the virology of recently emerged coronaviruses to gain an in-depth understanding of these infectious diseases.Conclusions: The presented information provides fundamental bottlenecks to devise future disease control and management strategies to curtail the impact of coronaviruses in human populations

Evolutionary analysis of infectious bronchitis virus reveals marked genetic diversity and recombination events

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 (n = 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

Co-circulation of avian influenza viruses in commercial farms, backyards and

Cloacal and tracheal swab-samples were collected from commercial farms, backyards and live market birds (LBM) to identify the potential existence and genetic drifts of avian influenza subtypes (AI) H5 and H9 that are circulating among bird species in Egypt. The results revealed that, one sample out of 50 samples of chicken commercial farms was positive for the isolation of subtype H9N2 [KC699549, Influenza A virus: A/chicken/Egypt/VRLCU-R33/2012(H9N2)]; from Sharkeia province. Two samples out of 20 samples of Backyard ducks were positive for the isolation of 2 subtypes H5N1; [KC699547, Influenza A virus: A/duck/Egypt/VRLCU-R11/2012(H5N1), “backyard duck”] from El-Fayoum province and the other from Giza province [A/duck/Egypt/VRLCU-R28/2012(H5N1), “backyard duck”]. Analysis of haemagglutinin (HA) and the phylogenetic tree of the isolated viruses (H5N1) were fallen within the clade 2.2.1.1. Antigenic cartography for the isolated Egyptian H9N2 AI virus can intuitively be of group-B. The number of mutations in the amino acid sites (33, 47, 65, 90, 92, 143, and 150) and the Long Branch observed in the phylogenetic tree may suggest a rather long evolution period. The sequenced H9N2 Egyptian virus in the study was closely related to the previous Egyptian isolates