Rania F. El Naggar

The genus Hepacivirus (HCV) has long been a dynamic group, increasing its number by myriads of species collectively referred to as non-primate hepaciviruses (NPHVs). NPHV exhibits a broad hepatotropism and is often attributed to chronic infection in horses and dogs. However, recent studies and meta-analyses on NPHV in horses have remained inconclusive regarding the determination of risk factors for infection. Therefore, our main goal was to investigate the frequencies and molecular characteristics of NPHV infection linked to geographical location, horse breeds, genders, and ages. For this purpose, we tested the positivity of 152 samples collected from ten cities in Turkey by conventional PCR, targeting the highly conserved 5′ UTR sequence, and compiled our results with pedigree data of horses for statistical analyses. We further implemented sequencing of the 5′ UTR, NS3, and NS5B regions and used in silico approaches to evaluate the characteristics of our novel isolates. Our results revealed a high incidence of NPHV infection (52.3%), which fluctuated among cities (26.1–75.0%), and comparative statistical analyses indicated that age and geographical region, together with managemental alterations, could be key determinants in NPHV infection. Furthermore, our phylogenetic analyses and 3D modelling approaches on genomic sequences revealed fundamental characteristics of novel NPHV strains. In conclusion, our study contributed to providing a better understanding of NPHV distribution and revealed some hints on diagnostics and good practices for disease prevention.

Members of the Anelloviridae family are increasingly being recognized for their role in veterinary and public health, with domestic cats identified as potential carriers of anelloviruses and gyroviruses. This study aimed to investigate the prevalence and genetic characteristics of these viruses in diarrheic cats from Sivas, Türkiye. A total of 91 fecal samples were analysed, initially for feline panleukopenia virus using conventional PCR, followed by screening with our Anelloviridae panel. The results revealed that 19 (20.9%) samples were positive for TTFeV1, 32 (35.2%) for CAV, 67 (73.6%) for Avian gyrovirus 2, four (4.4%) for Gyrovirus 3, and three (3.3%) for Gyrovirus 4. Statistical analyses revealed frequent co-infections among parvoviruses, anelloviruses, and gyroviruses, with a significant association between Gyrovirus chickenanemia (CAV) and Gyrovirus galga1 (AvGyV2). Notably, Gyrovirus 4 (Gyrovirus homsa3) was identified in feline stool for the first time. Phylogenetic and genomic analyses, based on partial TATA box-ORF2 sequences for anelloviruses and VP1 sequences for gyroviruses, provided further insights into viral diversity. These findings expand current knowledge of anellovirus and gyrovirus circulation in feline populations, underscoring the importance of continued surveillance for feline and public health.

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.

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.

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.

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.

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.

Avian coronaviruses (ACoVs) are continuously evolving and causing serious economic consequences in the poultry industry and around the globe. Owing to their extensive genetic diversity and high mutation rates, controlling ACoVs has become a challenge. In this context, the potential contribution of wild birds in the disease dynamics, especially in domesticated birds, remains largely unknown. In the present study, five hundred fifty-seven (n = 557) cloacal/fecal swabs were collected from four different wild bird species from eight Egyptian governorates during 2016 and a total of fourteen positive isolates were used for phylodynamics and evolutionary analysis. Genetic relatedness based on spike (S1) gene demonstrated the clustering of majority of these isolates where nine isolates grouped within Egy/variant 2 (IS/885 genotype) and five isolates clustered within Egy/variant 1 (IS/1494/06 genotype). Interestingly, these isolates showed noticeable genetic diversity and were clustered distal to the previously characterized Egy/variant 1 and Egy/variant 2 in Egyptian commercial poultry. The S1 gene based comparison of nucleotide identity percentages revealed that all fourteen isolates reported in this study were genetically related to the variant GI-23 lineage with 92–100% identity. Taken together, our results demonstrate that ACoVs are circulating in Egyptian wild birds and highlight their possible contributions in the disease dynamics. The study also proposes that regular monitoring of the ACoVs in wild birds is required to effectively assess the role of wild birds in disease spread, and the emergence of ACoVs strains in the country.

Innate antiviral immunity establishes first line of defense against invading pathogens through sensing their molecular structures such as viral RNA. This antiviral potential of innate immunity is mainly attributed to a myriad of IFN-stimulated genes (ISGs). Amongst well-characterized ISGs, we have previously shown that antiviral potential of chicken IFN-induced proteins with tetratricopeptides repeats 5 (chIFIT5) is determined by its interaction potential with 5′ppp containing viral RNA. Here, we generated transgenic chickens using avian sarcoma-leukosis virus (RCAS)-based gene transfer system that constitutively and stably express chIFIT5. The transgenic chickens infected with clinical dose (EID₅₀ 10⁴ for HPAIV and 10⁵ EID₅₀ for vNDV) of high pathogenicity avian influenza virus (HPAIV; H5N1) or velogenic strain of Newcastle disease virus (vNDV; Genotype VII) showed marked resistance against infections. While transgenic chickens failed to sustain a lethal dose of these viruses (EID₅₀ 10⁵ for HPAIV and 10⁶ EID₅₀ for vNDV), a delayed and lower level of clinical disease and mortality, reduced virus shedding and tissue damage was observed compared to non-transgenic control chickens. These observations suggest that stable expression of chIFIT5 alone is potentially insufficient in providing sterile protection against these highly virulent viruses; however, it is sufficient to ameliorate the clinical outcome of these RNA viruses. These findings propose the potential of innate immune genes in conferring genetic resistance in chickens against highly pathogenic and zoonotic viral pathogens causing sever disease in both animals and humans.