Respiratory disease complex due to mixed viral infections in chicken in Jordan.

The global distribution of avian respiratory viruses highlights the need for effective surveillance programs and international collaboration to monitor viral circulation and implement timely control measures. In the current study, we aim to provide a comprehensive overview of avian respiratory viral infections in the poultry flocks in Jordan, focusing on the major viruses involved, their epidemiology, clinical manifestations, and evolution based on viroinformatics that will be helpful to improve the diagnostic methods, and control strategies including vaccines in the region. In this research, various poultry broiler groups in Jordan experiencing respiratory symptoms were tested for respiratory viral pathogens from January 2021 to February 2022. The mortality rates observed in the examined groups varied between 6% and 40%. The identified strains were authenticated using the RT-qPCR assay. Furthermore, they underwent in-depth characterisation through the sequencing of the complete spike (S1) gene for infectious bronchitis virus (IBV) and the haemagglutinin (HA) gene for avian influenza virus (AIV) subtype H9N2. Co-infection of IBV and AIV H9N2 viruses was detected through molecular analysis. The IBV strains showed affiliation with the variant groups GI-16 (3 strains) and GI-23 (9 strains) and exhibited numerous mutations. Meanwhile, H9N2 avian influenza viruses displayed various changes in amino acids within the HA gene, suggesting the influence of antibody-driven selection pressure. The phylogenetic analysis revealed that the H9N2 viruses identified in this investigation shared close genetic ties with EG3 (3 strains) and the Middle East group (ME1; 8 strains). These strains have been recently found in Jordan and nearby countries in the Middle East. Moreover, their HA genes exhibited similarities to viruses belonging to the G1-like lineage. In conclusion, avian respiratory viral infections remain a significant concern for the poultry industry, requiring constant vigilance and proactive measures to minimise their impact. Continued surveillance, robust diagnostic methods, effective vaccines, and international cooperation are essential components of a comprehensive approach to combat avian respiratory viral infections (AI, IBV, ND and ILT 'viruses) and safeguard avian health and global poultry production.

Avian sarcoma/leukosis virus (RCAS)-mediated over-expression of IFITM3 protects chicks from highly pathogenic avian influenza virus subtype H5N1.

Broad-spectrum antiviral activities of interferon-induced transmembrane proteins (IFITMs) are primarily attributed to in vitro inhibition of viral entry. Here, we used an avian sarcoma-leukosis virus (RCAS)-based gene transfer system and successfully generated chicks that constitutively express chicken IFITM3 (chIFITM3). The chIFITM3-overexpressing chicks showed significant protection and disease tolerance against highly pathogenic avian influenza virus (HPAIV) H5N1 (Clade 2.2.1.2). The chicks, overexpressing chIFITM3, also showed delayed onset of clinical symptoms, reduced viral shedding, and alleviated histopathologic alterations compared to control and challenged chicks. These findings highlight that overexpression of chIFITM3 provide a substantial defense against zoonotic H5N1 in vivo.

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.

Evolutionary Trajectories of Avian Avulaviruses and Vaccines Compatibilities in Poultry.

Newcastle disease virus (NDV) causes one of the highly infectious avian diseases in poultry leading to genuine financial misfortunes around the world. Recently, there has been an increasing trend in the number of ND-associated outbreaks in commercial Jordanian poultry flocks indicating a possible complex evolutionary dynamic of NDV infections in the country. To underpin the dynamics of circulating NDV strains and to assess the vaccine-escape potential, a total of 130 samples were collected from different poultry flocks in six Jordanian Governorates during 2019-2021. Twenty positive isolates, based on real-time reverse transcriptase PCR, were used for further genetic characterization and evolutionary analysis. Our results showed that there is a high evolutionary distance between the newly identified NDV strains (genotype VII.1.1) in this study and the commercially used vaccines (genotypes I and II), suggesting that circulating NDV field strains are under constant evolutionary pressure. These mutations may significantly affect flocks that have received vaccinations as well as flocks with insufficient immunity in terms of viral immunity and disease dynamics. To assess this further, we investigated the efficacy of the heterologous inactivated LaSota or homologous genotype VII.1.1 vaccine for their protection against virulent NDV in chicken. Vaccine-induced immunity was evaluated based on the serology, and protection efficacy was assessed based on clinical signs, survival rates, histopathology, and viral shedding. Chickens vaccinated with the inactivated genotype VII.1.1 based vaccine showed 100% protection with a significant reduction in virus shedding, and ameliorated histopathology lesions compared to LaSota vaccinated chicks that showed 60% protection. These results revealed that the usage of NDV inactivated vaccine from the circulating field strains can successfully ameliorate the clinical outcome and virus pathobiology in vaccinated chicks and will serve as an effective vaccine against the threat posed by commonly circulating NDV strains in the poultry industry.

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 sarcoma-leukosis 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.

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.

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.

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.

Comparative clinico-pathological assessment of velogenic (sub-genotype VIIi) and mesogenic (sub-genotype VIm) Avian avulavirus 1 in chickens and pigeons.

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects a wide range of avian species worldwide. Recently, several AAvVs of diverse genotypes have emerged with varying genomic and residue substitutions, and subsequent clinical impact on susceptible avian species. We assessed the clinico-pathological influence of two different AAvV 1 pathotypes [wild bird originated-velogenic strain (sub-genotype VIIi, MF437287) and feral pigeon originated-mesogenic strain (sub-genotype VIm, KU885949)] in commercial broiler chickens and pigeons. The velogenic strain caused 100% mortality in both avian species while the mesogenic strain caused 0% and 30% mortality in chickens and pigeons, respectively. Both strains showed tissue tropism for multiple tissues including visceral organs; however, minor variances were observed according to host and pathotype. The observed gross and microscopic lesions were typical of AAvV 1 infection. Utilizing oropharyngeal and cloacal swabs, a comparable pattern of viral shedding was observed for both strains from each of the infected individuals of both avian species. The study concludes a varying susceptibility of chickens and pigeons to different wild bird-originated AAvV 1 pathotypes and, therefore, suggests continuous monitoring and surveillance of currently prevailing strains for effective control of the disease worldwide, particularly in disease-endemic countries.

Emergence and genetic analysis of variant pathogenic 4/91 (serotype 793/B) infectious bronchitis virus in Egypt during 2019.

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.

Biological characterization of wild-bird-origin avian avulavirus 1 and efficacy of currently applied vaccines against potential infection in commercial poultry.

Newcastle disease virus (NDV), the type member of the species Avian avulavirus 1 (formerly known as avian paramyxovirus serotype 1), causes a highly contagious and economically important disease in a myriad of avian species around the globe. While extensive vaccination programs have been implemented in ND-endemic countries, the disease is continuously spreading in commercial, backyard, and wild captive poultry. In order to investigate the evolution of the virus and assess the efficiency of the vaccine regimens that are currently being applied in commercial poultry, four wild-bird-origin NDV strains were characterized biologically, based on mean death time and intracerebral pathogenicity index, and genetically, based on the cleavage motif (112RRQKRF117) in the fusion (F) protein. Based on these features, all of the isolates were characterized as velogenic strains of NDV. Phylogenetic analysis based on the complete genome sequence revealed clustering of these isolates within class II, genotype VII. This class of NDV remains the predominant genotype in the Egyptian poultry industry, as well as in those of many Asian and African countries. To investigate the potential of these wild-bird-origin NDV isolates to cause infection in domesticated poultry and to assess the efficacy of currently available vaccines for protection of commercial poultry, an extensive animal challenge experiment was performed. Cumulative clinicopathological and immunological investigations of virus-challenged chickens indicate that these isolates can potentially be transmitted between chicken and cause systemic infections, and the currently applied vaccines are unable to prevent clinical disease and virus shedding. Taken together, the data represent a comprehensive evaluation of the ability of Egyptian wild-bird-origin NDV strains to cause infection in commercial poultry and highlights the need for a continuous and large-scale surveillance as well as revised vaccine approaches. These integrated and multifaceted strategies would be crucial in any efforts to control and eradicate the disease globally.