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.

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.

Selenium nanoparticles enhance the efficacy of homologous vaccine against the highly pathogenic avian influenza H5N1 virus in chickens.

A proper vaccination against avian influenza viruses in chicken can significantly reduce the risk of human infection. Egypt has the highest number of recorded humans highly pathogenic avian influenza (HPAI)-H5N1 infections worldwide despite the widespread use of homologous vaccines in poultry. Enhancing H5N1 vaccine efficacy is ultimately required to better control HPAI-H5N1. The aim of this study is to boost chicken immunity by combined with inactivated HPAI-H5N1 with selenium nanoparticles (SeNPs). The chickens groups 1-3 were fed diets supplemented with SeNPs concentrations (0.25, 0.5, and 1 mg/kg) for 3 weeks and then vaccinated (inactivated HPAI-H5N1). while groups 4,5 and 6 were fed with SeNPs free diets and administered with 0.5 ml of the vaccine combined with 0.02, 0.06, and 0.1 mg/dose of SeNPs and then all groups were challenged with homologous virus 3 weeks post-vaccination (WPV). Group 7, 8 were used as control positive and negative respectively. At 4, 5, and 6 WPV, antibody titer was considerably higher in the group fed a meal supplemented with 1 mg SeNPs/kg. In contrast, both methods of SeNPs supplementation significantly increased the Interleukin 2 (IL2), Interleukin 6 (IL6), and Interferon γ (IFNγ) expressions in the blood cells in a dose-dependent manner, with a higher expression observed in the group that was vaccinated with 0.1 mg/dose. After the challenge, all groups that received SeNPs via diet or vaccines dose showed significant reduction in viral shedding and milder inflammation in lung, trachea, spleen, and liver in addition to higher expression of IL2, IL6, and IFNγ, with the highest expression observed in the group that was vaccinated with 0.1 mg/dose compared the plain vaccinated group. The groups of 1 mg SeNPs/kg and combined vaccinated with 0.1 mg/dose showed the best vaccine efficacy. However, the group vaccinated with 0.1 mg/dose showed the earliest reduction in viral shedding. Overall, SeNPs supplementation in the diet and the administration of the vaccine formula with SeNPs could enhance vaccine efficacy and provide better protection against HPAI-H5N1 in chickens by enhancing cellular immunity and reducing inflammation. We recommend using SeNPs as a vaccine combination or feeding with diet to increase the immunity and vaccine efficacy against H5N1.

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 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.

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.

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.