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Background: Newcastle disease virus (NDV) genotype VII severely affects poultry, causing respiratory and neurological symptoms with a high rate of morbidity and mortality. The research aimed to develop an inactivated ND vaccine using local isolates (Genotype VII.2) and assess its immunogenicity compared to other commercial live ND vaccines., Methods: An inactivated vaccine using a candidate NDV (GenBank: OR924274.1) was developed according to WOAH guidelines following inactivation, sterility, purity, and safety tests. The birds were vaccinated through subcutaneous (SC) and intramuscular (IM) routes using three doses (0.25, 0.5, and 1.0 ml/bird). Immunogenicity and protective potentiality of the experimentally developed inactivated ND vaccine and live commercial ND vaccine (intra-ocularly/IO) were compared by challenge studies using three vaccination schedules: killed-followed-killed, live-followed-killed, and live-followed-live., Results: The birds vaccinated with 1.0 ml/bird SC showed higher antibody titers compared to those of IM-vaccinated groups. Birds vaccinated with the live-followed-killed commercial ND vaccines had slightly higher antibody titers compared to those vaccinated with killed-followed-killed and live-followed-live vaccines. Birds vaccinated with the killed-followed-killed ND vaccine showed a higher protection rate (100%) compared to live-followed-killed (83±5.77%) and live-followed-live (57±5.77%) vaccines. Birds vaccinated with killed-followed-killed group showed a slower decline rate of antibody titers than other groups. This regimen provided significantly better immunity, highlighting its potential in controlling ND outbreaks in Bangladesh's poultry., Conclusion: The study found that the inactivated ND vaccine, developed with the locally circulating isolate of genotype-VII.2 of NDV, might play an important role in effective control and management of ND in the commercial poultry population in Bangladesh., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Haque, Haque, Parvin, Kamal, Islam, Sadekuzzaman, Islam, Khatun, Hossain, Uddin, Nahar, Khasruzzaman and Islam.)

Infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis (IB), a severe disease that primarily affects young chickens and poses a significant challenge to the global poultry industry. Understanding the complex interaction between the virus and its host is vital for developing innovative antiviral strategies. Long non-coding RNA (lncRNA) plays a crucial role in regulating host antiviral immune responses. Our previous studies have shown that IBV infection disrupts the stability of lncRNA in host cells, indicating a potential regulatory role for lncRNA in IBV pathogenesis. It is still not clear how lncRNA precisely modulates IBV replication. In this study, we observed down-regulation ofMSTRG.26120.58 (named lncRNA-DRNR) expression in various chicken cell lines upon IBV infection. We demonstrated that silencing lncRNA-DRNR using siRNA enhances intracellular replication of IBV. Through exploring genes encoding proteins upstream and downstream of lncRNA-DRNR within a 100 kb range, we identified chJMJD6 (chicken JMJD6) as a potential target gene negatively regulated by lncRNA-DRNR expression levels. Furthermore, chJMJD6 inhibits STAT1 methylation, thereby affecting the induction of interferon-stimulated genes (ISGs) through the activation of the IFN-β-mediated JAK-STAT signalling pathway, ultimately promoting the intracellular replication of IBV. In summary, our findings reveal the critical role played by lncRNA-DRNR during IBV infection, providing novel insights into mechanisms underlying coronavirus-induced disruption in lncRNA stability., (© 2024. The Author(s).)

Background: H9N2 avian influenza viruses have been circulating in Bangladesh since 2006, affecting multiple avian species and resulting in economic losses. The recent emergence of tribasic strains, along with co-infections, has increased the risk to poultry health. Therefore, the study aimed to compare the immune responses of Sonali (crossbred) and commercial broiler chickens infected with tribasic H9N2 low pathogenic avian influenza (LPAI) virus., Methods: Following H9N2 infection, proinflammatory (IL-6, IL-8, IL-1β and TNF-α) and antiviral (IFN-β and IFN-γ) cytokine expressions were observed in the trachea, lungs, intestine, and lymphoid tissues in Sonali and broiler chickens from 1 day post infection (dpi) to 10 dpi by qPCR., Results: Sonali chickens exhibited significantly higher proinflammatory and antiviral cytokine expressions in the trachea at 3-7 days post infection (dpi), while broiler chickens showed lower immune responses. Broiler chickens displayed prolonged IL-6, IL-8, and IL-1β expression in lungs at 3-10 dpi compared to Sonali chickens. In the intestine, broiler chickens showed higher IL-6 and IL-8 expression that peaks at 1-3 dpi, while in Sonali chickens only IL-1β elevated at 10 dpi. In response to the H9N2 viruses, broiler chickens exhibited a stronger early IFN-β responses and a delayed IFN-γ responses in their lymphoid organs compared to Sonali chickens., Conclusion: This suggests distinct immune profiles between the chicken types in response to the H9N2 infection. The information sheds light on the function of innate immunity in the pathophysiology of currently circulating tribasic H9N2 virus and could assist in effective controlling of avian influenza virus spread in poultry and designing vaccines., (© 2024. The Author(s).)

The guinea fowl (Numida meleagris) holds significant agricultural importance in Ghana, particularly in the Northern, Upper East and Upper West Regions. Despite their economic and cultural significance, guinea fowls face a potential threat from avian influenza, a global concern for its adverse impact on poultry populations. This study assessed the seroprevalence of the virus in mature guinea fowls in the Upper East Region. A cross-sectional survey was conducted in three districts within the Upper East Region from April to June 2023. Blood samples were collected from 397 guinea fowls that are over 4 weeks old, and seroprevalence was determined using ID Screen Influenza A Antibody Competition Enzyme-linked immunosorbent assay (ELISA). The study analysed demographic factors such as sex, age and source of birds, employing statistical methods to establish associations. Among the sampled guinea fowls, 24.7% tested positive for avian influenza antibodies, whereas 75.3% were seronegative. Age did not show statistically significant associations with seroprevalence, but intriguing patterns were observed. Adult guinea fowls exhibited higher seroprevalence (23.7%) compared to growers (1.0%). The source of birds showed no significant association, but birds from slaughter points demonstrated higher seroprevalence (11.6%) compared to households (5.0%) and live bird markets (8.1%). In conclusion, the study underscores the importance of monitoring avian influenza in guinea fowls to implement effective control measures. The presence of antibodies suggests guinea fowls may contribute to virus transmission in the Upper East Region. The study recommends ongoing nationwide surveillance to assess the true prevalence of avian influenza in guinea fowls across Ghana., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Vaccine adjuvants, serving as non-specific immune enhancers, play a pivotal role in the immunoprevention and control of animal diseases. This study utilized prokaryotic expression systems to express and purify chicken-derived cIFITM1, cIFITM3, and cViperin, which were then formulated as adjuvants with H9N2 avian influenza virus antigens to create inactivated vaccines. These vaccines were administered to SPF chickens to investigate their immunopotentiating functions. Additionally, the proteins were assessed for their ability to act as standalone immune enhancers. The results demonstrated that cIFITM1, cIFITM3, and cViperin significantly elevated serum hemagglutination inhibition (HI) antibody titers. Notably, when used individually, these proteins markedly enhanced the antiviral capabilities of challenged chickens, leading to alleviated clinical symptoms, reduced tracheal virus replication, diminished virus shedding, and lessened histopathological damage, with cIFITM1 exhibiting the most pronounced effect. Furthermore, the protective efficacy of two H9N2 recombinant virus inactivated vaccines supplemented with cIFITM1 adjuvant was validated, achieving a 100 % vaccine protection efficiency. In conclusion, cIFITM1, cIFITM3, and cViperin as adjuvants for influenza vaccines effectively inhibit virus replication and shedding, highlighting their significant potential in influenza prevention and control., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Duck hepatitis A virus (DHAV) infection in ducklings causes acute hepatitis with considerable economic losses. In this study, Pekin and Muscovy duckling flocks (n=9) suffering from high mortality and hepatic lesions were examined by RT-PCR for DVHA. 44.4 % (4/9) of samples were positive for DHAV (5' UTR region), of which 100 % (4/4) were DVHA-3 (VP1 gene). VP1 sequencing and phylogenetic analysis of an isolate originated from Muscovy ducklings showed that it shared 96.8 % -100 %, 88.5-89.2 %, and 86.5-88.2 % nucleotide similarity (ns) with the Egyptian, Korean-Vietnamese, and Chinese DVHA-3 strains, respectively. It was distinguished from the DHAV-1 vaccine (67.6 % ns). The sequenced DVHA-3 isolate was used to experimentally infect 5-day-old Pekin and Muscovy ducklings vs. control groups. No apparent clinical signs or deaths were reported in the experimentally-infected groups. Pekin ducklings showed greater cloacal viral shedding than Muscovy until the 6th dpi (P<0.05). DVHA-3 induced a significant rise in IFN-β and IL-1β serum levels, where Muscovy ducklings' levels were higher than Pekin ducklings. Among the biochemical parameters, AST was only increased on the 4th dpi in both breeds vs. control (P<0.05). Compared to Muscovy ducklings at 2, 4, and 6 day post infection (dpi), the infected Pekin group had lower lipase levels (P≥0.05, p<0.05, and p<0.05, respectively), while ALT was higher at 4 and 6 dpi (P<0.05). The histopathological lesions supported the gross lesions, and their scores were dominant at 2 and 4 dpi in both breeds. At 6 and 8 dpi, Pekin showed more severe histopathological changes compared to Muscovy for the liver, heart, brain, and intestines; the pancreas, kidney, and lung showed the opposite pattern. In conclusion, Pekin ducklings displayed distinct DHAV-3 infection results from Muscovy ducklings, and more research utilizing a variety of DHAV-3 strains has to be carried out., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The H9N2 inactivated avian influenza vaccine cannot induce cellular and mucosal immune responses, while the attenuated Salmonella vector as an intracellular bacterium can induce dominant cellular and mucosal immune responses. However, it provides low protection against the virus when delivering viral antigens and needs further optimization. Chicken C-C motif chemokine ligand 5 (chCCL5) is an important CC chemokine associated with immune cell chemotaxis, migration, and viral infection. This study connected the sequence of chCCL5 (CCL5) with the hemagglutinin sequence of the H9N2 avian influenza virus (yH9HA), utilizing the attenuated Salmonella typhimurium vector containing the delayed lysis system MazE/F regulated by arabinose as a carrier. A vaccine strain of recombinant attenuated Salmonella typhimurium and H9N2 avian influenza virus HA, rSC0130 (pS0017-yH9HA-CCL5), was successfully constructed. The experimental results indicate that yH9HA-CCL5 can be expressed in 293 T cells; compared to the strain without CCL5, rSC0130 (pS0017-yH9HA-CCL5) can induce significantly increased cellular immune responses and provide better protective effects in H9N2 virus challenge experiments. The above results indicate that chCCL5 can significantly enhance the protective effect of Salmonella delivering H9N2 avian influenza virus HA protein vaccine against H9N2 avian influenza virus infection, providing valuable theoretical support for further improving the protective efficiency of recombinant attenuated Salmonella vectors for delivering viral antigens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Orthoflaviviruses are single-stranded RNA viruses characterized by highly efficient self-amplification of RNA in host cells, which makes them attractive vehicles for vaccines. Numerous preclinical and clinical studies have demonstrated the efficacy and safety of orthoflavivirus replicon vectors for vaccine development. In this study, we constructed Tembusu virus (TMUV) replicon-based single-round infectious particles (SRIPs) as vaccine development platform. To evaluate the potential of TMUV SRIPs as vaccines, we generated SRIPs that express the heterologous Fowl adenovirus 4 (FAdV-4) fiber2 protein and fiber2 head domain, named TMUVRP-fiber2 and TMUVRP-fiber2H, respectively. To assess the immunogenicity of the TMUV SRIPs, SPF chicks were intramuscularly inoculated twice. Our results showed that the TMUVRP-fiber2 vaccines elicited high levels of neutralizing antibodies. Challenge experiments showed that TMUVRP-fiber2 provided full protection against virulent FAdV-4 and significantly reduced viral shedding. Moreover, the immunogenicity of TMUVRP-fiber2H was significantly lower than that of TMUVRP-fiber2, which was reflected in the neutralizing antibody titer, survival rate, and virus shedding after challenge. Therefore, our results suggested that TMUV SRIPs are a promising novel platform for the development of vaccines for existing and emerging poultry diseases., Competing Interests: Declaration of Competing Interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024. Published by Elsevier B.V.)

Inclusion body hepatitis (IBH) induced by fowl adenovirus serotype 8b (FAdV-8b) infection is an important avian infectious disease circulating around the globe, posing significant losses to the poultry industry. In this study, a FAdV-8b strain, CH/SDQD/2021, was isolated from IBH-affected chickens in Shandong province, China and the genetic properties of CH/SDQD/2021 were characterized. The full genome length of CH/SDQD/2021 is 44,000 bp, with a G+C content of 58 % and 32 open reading frames (ORF). Sequencing alignment and phylogenetic analysis indicated that the genome identity of CH/SDQD/2021 compared to 30 other FAdV-E strains retrieved from GenBank ranges from 89.72 % to 96.71 %. Animal regression test indicated that CH/SDQD/2021 infection induced IBH in one-week-old SPF chickens. Subsequently, a reverse genetic system was developed to facilitate rapid genome manipulation of FAdV-8b for gene function study and vaccine development. To explore potential foreign gene insertion sites in FAdV-8b, ORF0-1-2, ORF11 and ORF19 of CH/SDQD/2021 were substituted by the green fluorescent gene ZsGreen, respectively, and the corresponding recombinant viruses were successfully rescued. The results showed that comparing with the parental FAdV-8b, the replication efficiency of the ORF0-1-2-substituted recombinant was reduced, while the replication efficiency of the ORF11-substituted recombinant was promoted. The findings of this study enrich the epidemiological data for the prevalent FAdV strains in China. Furthermore, the establishment of the FAdV-8b reverse genetic system will provide an efficient technique platform for FAdV-8b gene function research at the whole virus level and developing related multivalent vaccine candidates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Avibirnavirus, specifically Infectious Bursal Disease Virus (IBDV), is a highly contagious pathogen that causes significant economic losses in the poultry industry. The polymerase protein VP1 of IBDV is critical to the viral life cycle, facilitating the synthesis of viral mRNA and the genome. Previous studies have suggested that various host factors influence the regulation of IBDV polymerase activity. In this study, we identified that IBDV infection induces the expression of optineurin (OPTN), a mitophagy receptor and a protein associated with amyotrophic lateral sclerosis (ALS), as well as a negative regulator of interferon I production. The induced expression of OPTN acts as a suppressor of IBDV replication, a function dependent on its ubiquitin-binding domain (UBAN). Furthermore, we demonstrated that OPTN exerts its antiviral effects through direct interactions with VP1 and VP3, which inhibit the polymerase activity of VP1 by preventing K63-linked ubiquitination of VP1. To our knowledge, this study is the first to report that OPTN, upregulated during IBDV infection, functions as a novel antiviral host factor that limits the virus's replicative capacity, offering a potential target for anti-IBDV therapeutic strategies., Competing Interests: Declaration of Competing Interest All of the authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Avian leukosis virus subgroup J (ALV-J) primarily affects poultry, particularly chickens, leading to tumourigenesis and immunosuppression, which results in substantial economic losses. It is important to note that ALV-J is commonly found in indigenous chicken breeds in China, and the virus's vertical transmission characteristics present a significant threat to the preservation of local chicken breeds., Objectives: The study aimed to investigate the characteristics and effects of the recombinant ALV-J strain LY2021J, with a focus on its genetic composition and its potential influence on virulence and pathogenicity., Methods: LY2021J was isolated using DF-1 cells and validated by enzyme-linked immunosorbent assay (ELISA) and IFA. The proviral genome was amplified using segmented PCR and then spliced together using DNASTAR software. Genome-wide genes, including gag, pol, gp85, and long terminal repeat (LTR), were compared. Recombination sites were analysed using RDP5 and SimPlot software. Pathogenicity was evaluated by monitoring symptoms and conducting examinations on SPF chickens., Results: The outbreak of ALV-J in China has caused significant economic losses in the poultry industry. Although largely controlled in white-feather broilers and egg-laying chickens, ALV-J has spread to yellow-feather broilers and local breeds. A strain, LY2021J, isolated from Hetian chickens, showed lower mortality despite severe dysplasia. Genetic analysis revealed high similarity between LY2021J and the Chinese strains JS14NT01 and NX0101, suggesting a shared origin. Recombination with strain ev-1 and specific 3' UTR deletions may explain LY2021J's reduced virulence. Continued monitoring and prevention strategies are essential to mitigate ALV-J's impact., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Avian leukosis is a significant viral disease affecting chicken populations globally, including Bangladesh, resulting in high mortality and morbidity rates and causing substantial economic losses in the commercial poultry industry. This study aimed to detect avian leukosis virus (ALV) during recent outbreaks in Bangladesh, utilising a molecular-based approach. A total of 14 liver samples were collected from the suspected layer flocks in Bangladesh. The diagnosis of ALV infection in chickens was confirmed through necropsy, histopathological examinations, reverse transcription-polymerase chain reaction (RT-PCR), and sequence analysis. Gross observations revealed severe liver enlargement with scattered white nodules on the surface in the infected chickens. Histopathological observations showed the infiltration of huge mononuclear inflammatory cells in the periportal area of liver and microvesicular fatty degeneration and necrosis of some hepatocytes. RT-PCR results identified three samples positive for the env gene of ALV. Sequence analysis of the env genes demonstrated high homology among the identified strains (97%-98%) and with reference strains (92%-96%) at the nucleotide level. The phylogenetic tree revealed close relatedness of the three identified strains to reference strains from India, USA, and China. Mutational analysis indicated several mutations throughout the envelope glycoprotein of the identified strains. Protein structure analysis showed minor changes in the hydrophobic region of the envelope protein of the identified strains. In conclusion, this study, the first detailed investigation in Bangladesh, contributes to understanding ALV epidemiology, highlights genetic diversity, and emphasises the necessity for further investigations and the implementation of effective control measures in the affected regions., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The H9N2 subtype of the avian influenza virus (AIV) poses a significant threat to the poultry industry and human health. Recombinant vaccines are the preferred method of controlling H9N2 AIV, and Marek's disease virus (MDV) is the ideal vector for recombinant vaccines. During this study, we constructed two recombinant MDV type 1 strains that carry the hemagglutinin (HA) gene of AIV to provide dual protection against both AIV and MDV. To assess the effects of different MDV insertion sites on the protective efficacy of H9N2 AIV, the HA gene of H9N2 AIV was inserted in UL41 and US2 of the MDV type 1 vector backbone to obtain recombinant viruses rMDV-UL41/HA and rMDV-US2/HA, respectively. An indirect immunofluorescence assay showed sustained expression of HA protein in both recombinant viruses. Additionally, the insertion of the HA gene in UL41 and US2 did not affect MDV replication in cell cultures. After immunization of specific pathogen-free chickens, although both the rMDV-UL41/HA and rMDV-US2/HA groups exhibited similar levels of hemagglutination inhibition antibody titers, only the rMDV-UL41/HA group provided complete protection against the H9N2 AIV challenge, and also offered complete protection against challenge with MDV. These results demonstrated that rMDV-UL41/HA could be used as a promising bivalent vaccine strain against both H9N2 avian influenza and Marek's disease in chickens., Competing Interests: Declaration of Competing Interest The authors of this study have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Avian hepatitis E virus (HEV) has resulted in significant economic losses in the poultry industry. There is currently no commercial vaccination available to prevent avian HEV infection. Previously, a novel epitope ( 601 TFPS 604 ) was discovered in the ORF2 protein of avian HEV. In this study, peptides were synthesized and assessed for their ability to provide immunoprotecting against avian HEV infection in poultry. Twenty-five Hy-Line Variety Brown laying hens were randomly divided into five groups; groups 1 to 3 respectively immunized with RLLDRLSRTFPS, PETRRLLDRLSR (irrelevant peptide control), or truncated avian HEV ORF2 protein (aa 339-606), while group 4 (negative control) was mock-immunized with PBS and group 5 (normal control) was not immunized or challenged. After the challenge, all hens in groups 2 and 4 showed seroconversion, fecal virus shedding, viremia, alanine aminotransferase (ALT) level increasing, liver lesions and HEV antigen in the liver. There were no pathogenic effects in other groups. Collectively, all of these findings showed that hens were completely protected against avian HEV infection when they were immunized with the peptide containing TFPS of the avian HEV ORF2 protein., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Salmonella is a globally prevalent foodborne pathogen, and adverse events caused by S. Enteritidis and S. Typhimurium are extremely common. With the emergence of drug resistance, there is an urgent need for efficient and specific lytic bacteriophages as alternative to antibiotics in clinical practice. In this study, phage P6 was isolated and screened from effluent and fecal samples from duck farm environments to specifically lyse the duck sources S. Typhimurium and S. Enteritidis. Phage P6 belongs to the genus Lederbergvirus, unclassified Lederbergvirus species. The phage P6 genome did not contained non-coding RNA, virulence genes and drug resistance genes, indicating that phage P6 was biologically safe for clinical applications. Phage P6 lysed 77.78% (28/36) of multidrug-resistant Salmonella and reduced biofilms formed by S. Enteritidis CVCC 3377, 4, and 24, and S. Typhimurium 44 by 44% to 75% within 3 h, and decreased Salmonella in duckling feces by up to 1.64 orders of magnitude. Prokaryotic expression of endolysin LysP6 lysed the chloroform-treated bacterial outer membrane from different serotypes of duck-derived Salmonella and E. coli standard strain ATCC 25922. The host range was expanded compared to phage P6, and the growth of Salmonella was effectively inhibited by LysP6 in conjunction with the membrane permeabilizer EDTA within 24 h. Therefore, phage P6 and phage-derived endolysins LysP6 are suitable for application as potent biocontrol agents to improve poultry health and food safety., Competing Interests: DISCLOSURES The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study investigated the diet-additive interactions of a Lactobacilli-based probiotic (Pro) and postbiotic (Post) on immune parameters and cecal microbiota composition, with subsequent effects on the metabolome in broilers. A completely randomized block design was employed with 2 diets [standard (SD), and challenge (CD)] and 3 additive conditions (Control, Pro, Post) involving 1,368 one-day-old male Ross 308 broilers equally distributed among 36 pens in a 42 d study. Diets were formulated to contain identical nutrient levels, with CD higher than SD in non-starch polysaccharide content by including rye and barley. Total non-specific serum Ig A, M and G concentrations were determined weekly from d14 to 35. Following vaccination, titres of specific antibodies binding Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) were measured. Microbiota composition was analyzed by 16S rRNA gene sequencing at d14 and 35, and α- and β-diversity indexes (Observed, Chao1, Bray, Jaccard) were calculated. Cecal short-chain fatty acids and the semi-polar metabolome were determined in the Control SD and all CD groups at d35. At d35, a diet-additive interaction was observed on cecal microbiota composition. Within SD, Pro and Post did not affect operational taxonomic units (OTU) abundance (adjusted-P > 0.05) and diversity indexes (P > 0.05). Within CD, Pro and Post affected the relative abundances of 37 and 44 OTUs, respectively (adjusted-P < 0.05), with Post but not Pro affecting β-diversity indexes (P = 0.041 and 0.064 for Bray and Jaccard, respectively). Within CD, Post increased cecal acetate (21%; P = 0.007) and butyrate (41%; P = 0.002) concentration and affected the concentration of 2 metabolites (adjusted-P < 0.05), while Pro affected 240 metabolites (adjusted-P < 0.05). No diet-additive interactions were observed on serum Ig (P > 0.05), except for IgM at d14 (P = 0.004). Diet composition, but not the additives, affected immune status parameters. The Pro and Post affected cecal microbiota composition only under dietary challenging conditions as previously reported for growth., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Coronaviruses (CoVs) are significant animal and human pathogens, characterized by being enveloped RNA viruses with positive-sense single-stranded RNA. The Coronaviridae family encompasses four genera, among which gammacoronaviruses pose a major threat to the poultry industry, which infectious bronchitis virus (IBV) being the most prominent of these threats. Particularly, IBV adversely affects broiler growth and egg production, causing substantial losses. The IBV strains currently circulating in Taiwan include the IBV Taiwan-I (TW-I) serotype, IBV Taiwan-II (TW-II) serotype, and vaccine strains. Therefore, ongoing efforts have focused on developing novel vaccines and discovering antiviral agents. The envelope (E) proteins of CoVs accumulate in the endoplasmic reticulum-Golgi intermediate compartment prior to virus budding. These E proteins assemble into viroporins, exhibiting ion channel activity that leads to cell membrane disruption, making them attractive targets for antiviral therapy. In this study, we investigated the E proteins of IBV H-120, as well as IBV serotypes TW-I and TW-II. E protein expression resulted in inhibited bacteria growth, increased permeability of bacteria to β-galactosidase substrates, and blocked protein synthesis of bacteria by hygromycin B (HygB). Furthermore, in the presence of E proteins, HygB also impeded protein translation in DF-1 cells and damaged their membrane integrity. Collectively, these findings confirm the viroporin activity of the E proteins from IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. Next, the viroporin inhibitors, 5-(N,N-hexamethylene) amiloride (HMA) and 4,4'-diisothiocyano stilbene-2,2'-disulphonic acid (DIDS) were used to inhibit the viroporin activities of the E proteins of IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. In chicken embryos and chickens infected with IBV serotypes TW-I and IBV TW-II, no survivors were observed at 6 and 11 days post-infection (dpi), respectively. However, treatments with both DIDS and HMA increased the survival rates in infected chicken embryos and chickens and mitigated histopathological lesions in the trachea and kidney. Additionally, a 3D pentameric structure of the IBV E protein was constructed via homology modeling. As expected, both inhibitors were found to bind to the lipid-facing surface within the transmembrane domain of the E protein, inhibiting ion conduction. Taken together, our findings provide comprehensive evidence supporting the use of viroporin inhibitors as promising antiviral agents against IBV Taiwan isolates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The clinical relevance of avian metapneumovirus (aMPV) is growing in the poultry sector, especially in broiler farming, where no vaccination is administered in Italy. Given the naïve status of the birds, a serological survey was conducted in a densely populated area of Northern Italy, to evaluate aMPV circulation. Seven farms were selected and sampled in summer/fall, then sampling was repeated in the following season (winter/spring) to assess a possible seasonal effect. In each farm, fifteen birds were blood sampled towards the end of the cycle and sera were analyzed with an ELISA test. Clinical signs were reported in 5 out of 7 farms, although all farms were positive at both sampling points, except for one, which was negative at the first sampling. The seroprevalence within farm ranged from 26.6% to 100%, and antibody titres appear to increase with age. No seasonality effect was evidenced, whereas a farm effect was more distinct. aMPV circulation appears wide in Northern Italian farms, with different clinical outcomes that could be modulated by intrinsic characteristics of the farms. In absence of vaccination, serological monitoring can be a useful tool for viral entrance monitoring, although sampling timing should be evaluated in order to spot seroconversion after late infections., Competing Interests: DISCLOSURES The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian reovirus (ARV) is the causative agent of avian viral arthritis and causes significant economic losses to the global poultry industry. For clinical diagnosis, detecting ARV-specific antibodies is crucial. We successfully expressed the ARV-σC protein in insect cells using the baculovirus expression vector system, achieving an expression level of approximately 200 mg/L. We developed an indirect enzyme-linked immunosorbent assay (iELISA) using the ARV-σC protein as a coating antigen to detect antibodies against it. The inter-batch and intrabatch coefficients of iELISA variation were less than 10%. Its sensitivity (1:12,800 diluted in serum) was 4 times higher than that of the indirect immunofluorescence assay (IFA; 1:3200 diluted in serum), and it showed no cross-reactivity with antibodies against other common avian viruses (such as Infectious bursal disease virus, Newcastle disease virus). The practicality of the iELISA was further evaluated using clinical samples. 300 clinical sera from chickens vaccinated with the ARV attenuated vaccine and 20 SPF sera were tested using both the iELISA and the IFA, demonstrating a 100% conformity rate. In conclusion, these results suggest that the iELISA developed in this study is a rapid, sensitive, and specific method that could serve as an effective diagnostic tool for monitoring and controlling avian viral arthritis., Competing Interests: DISCLOSURES All authors disclosed no relevant relationships., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Gyrovirus galga1 (GyVg1), formerly known as AGV2, was initially identified in chickens in southern Brazil. The prevalence of GyVg1 from 2021 to 2024 in 28 out of the 63 poultry farms located in Jiangsu, Anhui, Henan, Hunan, Shandong, and Hubei provinces in eastern and central China was detected via PCR. The complete genomes of the 28 strains were sequenced and exhibited a full length of 2,376 bp. Similarity analysis of these strains did not suggest definite correlation with evolutionary branching and geographical distribution. Compared with the reference GyVg1 strains, HN2202 shared the highest similarity of 99.71% with HLJ1511 (chicken-originated) from northeastern China in 2015 to 2016. Recombination analysis revealed that AH2102 was a potential recombinant of peafowl-originated HN2019-PF1 and chicken-originated HLJ1506-2, whereas HN2304 was a recombinant of peafowl-originated HN2019-PF1 and the Hungarian ferret strain G13. Mutation site analysis of the capsid protein revealed that highly mutated regions occurred between sites 288 to 316 and 383 to 419. These results indicate that GyVg1 may have undergone an interspecies transmission, which involved complex mutations and recombination. This study may provide a reference for subsequent investigations targeting the molecular epidemiology and viral evolution of GyVg1., Competing Interests: DISCLOSURES The authors have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

From 2014-2023, infectious bronchitis virus (IBV) was detected in 6,589 samples from Canada, and partial nucleotide (nt) sequences of the IBV spike protein ( S ) gene were determined for 1,678 samples. Based on their S gene nt sequence identities and origin, Canadian IBVs could be classified into 4 groups: 1) 50.3% were variant viruses related to strains described in the United States; 2) 45.6% were vaccine-like viruses; 3) 2.1% were Eurasian viruses; 4) 2.0% were Canadian variants. Outbreaks with IBVs related to strains CAL1734/04, 4/91, and DMV/1639/11 were often associated with more severe disease in all chicken commodity groups. With the emergence of numerous IBV strains, the severity of infection and number of affected flocks increased. Outbreaks with various IBV strains overlapped in their emergence, peaked, and regressed, but the introduction of DMV/1639/11 has resulted in a continuous field challenge since its first detection in 2015., Competing Interests: Declaration of conflicting interestsWe declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Since its first isolation from migratory birds in Egypt in 2016, highly pathogenic avian influenza (HPAI) H5N8 has caused several outbreaks among domestic poultry in various areas of the country affecting poultry health and production systems. However, the genetic and biological properties of the H5N8 HPAI viruses have not been fully elucidated yet. In this study, we aimed to monitor the evolution of circulating H5N8 viruses and identify the pathogenicity and mammalian adaptation in vitro and in vivo. Three H5N8 HPAI viruses were used in this study and were isolated in 2021-2022 from poultry and wild birds during our routine surveillance. RNA extracts were subjected to full genome sequencing. Genetic, phylogenetic, and antigenic analyses were performed to assess viral characteristics and similarities to previously isolated viruses. Phylogenetic analysis showed that the hemagglutinin genes of the three isolates belonged to clade 2.3.4.4b and grouped with the 2019 viruses from G3 with high similarity to Russian and European lineages. Multiple basic amino acids were observed at cleavage sites in the hemagglutinin proteins of the H5N8 isolates, indicating high pathogenicity. In addition, several mutations associated with increased virulence and polymerase activity in mammals were observed. Growth kinetics assays showed that the H5N8 isolate is capable of replicating efficiently in mammalian cells lines. In vivo studies were conducted in SPF chickens (White Leghorn), mice, and hamsters to compare the virological characteristics of the 2022 H5N8 isolates with previous H5N8 viruses isolated in 2016 from the first introduction. The H5N8 viruses caused lethal infection in all tested chickens and transmitted by direct contact. However, we showed that the 2016 H5N8 virus causes a higher mortality in chickens compared to 2022 H5N8 virus. Moreover, the 2022 virus can replicate efficiently in hamsters and mice without preadaptation causing systemic infection. These findings underscore the need for continued surveillance of H5 viruses to identify circulating strains, determine the commercial vaccine's effectiveness, and identify zoonotic potential., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Moatasim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A/goose/Guangdong/1/96-like (GsGd) highly pathogenic avian influenza (HPAI) H5 viruses cause severe outbreaks in poultry when introduced. Since emergence in 1996, control measures in most countries have suppressed local GsGd transmission following introductions, making persistent transmission in domestic birds rare. However, geographical expansion of clade 2.3.4.4 sublineages has raised concern about establishment of endemic circulation, while mechanistic drivers leading to endemicity remain unknown. We reconstructed the evolutionary history of GsGd sublineage, clade 2.3.4.4c, in Taiwan using a time-heterogeneous rate phylogeographic model. During Taiwan's initial epidemic wave (January 2015 - August 2016), we inferred that localised outbreaks had multiple origins from rapid spread between counties/cities nationwide. Subsequently, outbreaks predominantly originated from a single county, Yunlin, where persistent transmission harbours the trunk viruses of the sublineage. Endemic hotspots determined by phylogeographic reconstruction largely predicted the locations of re-emerging outbreaks in Yunlin. The transition to endemicity involved a shift to chicken-dominant circulation, following the initial bidirectional spread between chicken and domestic waterfowl. Our results suggest that following their emergence in Taiwan, source-sink dynamics from a single county have maintained GsGd endemicity up until 2023, pointing to where control efforts should be targeted to eliminate the disease., (© 2024. The Author(s).)

Avian influenza viruses (AIVs) are a major economic burden to the poultry industry and pose serious zoonotic risks, with human infections being reported every year. To date, the vaccination of birds remains the most important method for the prevention and control of AIV outbreaks. Most national vaccination strategies against AIV infection use whole virus-inactivated vaccines, which predominantly trigger a systemic antibody-mediated immune response. There are currently no studies that have examined the antibody repertoire of birds that were infected with and/or vaccinated against AIV. To this end, we evaluate the changes in the H9N2-specific IgM and IgY repertoires in chickens subjected to vaccination(s) and/or infectious challenge. We show that a large proportion of the IgM and IgY clones were shared across multiple individuals, and these public clonal responses are dependent on both the immunisation status of the birds and the specific tissue that was examined. Furthermore, the analysis revealed specific clonal expansions that are restricted to particular H9N2 immunisation regimes. These results indicate that both the nature and number of immunisations are important drivers of the antibody responses and repertoire profiles in chickens following H9N2 antigenic stimulation. We discuss how the repertoire biology of avian B-cell responses may affect the success of AIV vaccination in chickens, in particular the implications of public versus private clonal selection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dascalu, Sealy, Sadeyen, Flammer, Fiddaman, Preston, Dixon, Bonsall, Smith and Iqbal.)

Avian metapneumovirus (aMPV) poses a significant global threat to the respiratory and reproductive systems of poultry, yet its prevalence and impact in specific regions, such as northern Thailand, remain understudied. This study is the first comprehensive assessment of aMPV seroprevalence among layer hens in the region. By the application of Enzyme-Linked Immunosorbent Assay (ELISA), our findings reveal a high overall seroprevalence of 60.78%, with significantly higher rates among post-laying hens (70.87%) compared to pre-laying hens (47.02%). Regional variations were noted, Uttaradit province showing the highest seroprevalence at 74%. Factors such as overcrowded conditions and poor farm hygiene were implied as contributors to increased seropositivity. Comparison with global studies highlights aMPV's endemicity and emphasizes the need for targeted surveillance and biosecurity measures. Migratory birds were inferred as potential vectors, contributing to the spread of aMPV across different regions. This study provides crucial epidemiological data essential for optimizing vaccination strategies and enhancing disease management practices to safeguard poultry health and sustainable production in northern Thailand., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: This paper reports the first pathological and molecular characterization of the novel variant of infectious bronchitis virus (IBV) D181 in poultry flocks in Morocco and Africa., Methods: The study includes six poultry farms, involving three flocks of layers aged between 28 and 67 weeks and three broiler flocks aged 27, 39 and 42 days from different regions of Morocco. In all affected layer flocks, a severe drop in egg production with poor eggshell quality was reported. Necropsy of dead birds was carried out, and samples of trachea, lungs, oviduct, ovaries, and kidneys were fixed in 10% neutral buffered formalin for histopathologic examinations, while other portions were stored at -20 °C for molecular analysis. Real time RT-qPCR for IBV gene group was performed, and IBV variants were identified. Partial S1 gene sequences were amplified by conventional RT-PCR, sequenced, and aligned for phylogenetic and amino acid similarity analysis., Results: Necropsy of dead birds revealed misshapen and hemorrhagic ovarian follicles with an edematous oviduct and severe reaction in the cecal tonsils. A caseous material accumulation in the sinus was noted in few birds. In contrast, the broiler flocks exhibited respiratory clinical signs such as difficulty in breathing, sneezing, tracheal rales, watery eyes and lethargy, associated with a decrease in feed consumption. Mortality in broiler ranged from 2 to 15%. Histopathological analysis of samples showed a lympho-plasmocytic inflammation in the oviduct, trachea, and lungs. Individual necrosis of epithelial cells, with sloughing of the bronchial epithelium and accumulation of desquamated cells with mucus in the airways, was observed in some birds. Partial S1 gene sequencing and phylogenetic analyses showed that the Moroccan strains were very closely related to D181 strains isolated in Dutch layers and breeders in 2018. Nucleotide sequence identities reached 90.9-95% with the Dutch isolates (strain CK/NL/D181/2018)., Conclusion: Our sequencing results demonstrate for the first time that the D181 IBV genotype is circulating in Moroccan poultry. These findings justify permanent monitoring of circulating strains in order to appropriately adjust vaccination strategies to align with the evolving field situation., (© 2024. The Author(s).)

Duck plague virus (DPV) causes the highly pathogenic duck plague, and the envelope glycoprotein I (gI), as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. This study used reverse genetics technology to target the gI, specifically within the DPV genome. Four DPV mutants with gI N-glycosylation site mutations were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues of gI (N 69 , N 78 , and N 265 ) are N-glycosylation sites, and western blot analysis substantiated that glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI leads to the protein misfolding and subsequent retention in the endoplasmic reticulum (ER). The subsequent deglycosylated gI is carried into the Golgi apparatus (GM130) in the interaction of gE. Compared to the parental virus, the mutated virus shows a 66.3% reduction in intercellular transmission capability. In ducks, the deglycosylation of gI significantly reduces DPV replication in vivo, thereby weakening the virulence of DPV. This study represents the first successful creation of a weak DPV virus strain by specific mutation at the N-glycosylation site. The findings provide a foundational understanding of DPV pathogenesis and form the basis for developing live attenuated vaccines against the disease., (© 2024. The Author(s).)

Infectious laryngotracheitis virus (ILTV) causes an acute and highly contagious respiratory disease in poultry. Live-attenuated vaccines are generally used to control and prevent infectious laryngotracheitis (ILT). However, these vaccines can revert to a virulent form due to multiple passages and thereby become an ILT source. Hence, monitoring of ILTV in the field through molecular characterization is critically important for controlling infection and differentiating circulating isolates. In this study, we genotypically characterized and phylogenetically analyzed eight ILTV isolates from chicken flocks located in four different cities of Turkey between 2019 and 2022. For all isolates, we analyzed two regions of the infected cell protein 4 gene (ICP4-1 and ICP4-2) and the thymidine kinase (TK) gene. The isolates were 100%, 100%, and 99.8-100% identical to each other in the ICP4-1 and ICP4-2 gene fragments and the TK gene, respectively. None of the ICP4 sequences had a deletion at nt 272-283, confirming that they were field isolates. None of the isolates were predicted to have a T252M mutation in the thymidine kinase, suggesting that they have low virulence. The isolates were 100%, 99.36%, and 99.91% identical to Turkish ILTV isolates in their ICP4-1, ICP4-2, and TK gene region, respectively. Phylogenetic analysis based on the ICP4-1 and TK genes confirmed that the ILTV isolates are closely related to Turkish ILTV isolates. This suggests that these ILTVs were endemic isolates, which in turn suggests that the ILTV isolates circulating in Turkey were evolutionarily close, originated from the field, and had low virulence., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

How landscape composition and configuration impact the distribution of multi-vector and multi-host mosquito vector-borne disease systems, such as West Nile virus (WNV), remains challenging because of complex habitat and resource requirements by hosts and vectors that affect transmission opportunities. We examined correlations between landscape composition and configuration and 2018 WNV sentinel chicken seroconversion in Florida, USA across the state and within five National Oceanic Atmospheric Administration (NOAA) bioclimatic regions to understand strength and variation of landscape effects during an elevated transmission year. Although few landscape studies have examined WNV in Florida, we expected higher percentages of residential or medium-developed landscapes and more fragmented landscapes would be positively correlated with WNV seroconversion owing to the main mosquito vector habitats and avian host distributions. However, we expected to find variation in the importance of forest, wetland, and agriculture landscapes across bioclimatic regions in the state. WNV seroconversion rates were calculated using Florida 2018 Department of Health WNV sentinel chicken seroconversion data from 187 flocks maintained by mosquito control programs. Percent land cover and edge density metrics were calculated for multiple land cover classes and within multiple buffer distances from chicken coops using 2019 National Land Cover Data. We used binomial generalized linear mixed effects models to calculate the importance of landscape metrics to WNV seroconversion. We found no statewide predictors of seroconversion, but as expected, the importance of landscape varied across regions. In the north-central part of the state, we found higher seroconversion in less populated suburban areas while higher seroconversion in south-central Florida was correlated with fragmented forested areas within 0.5 km of coops and intact woody wetland areas within 2 km of coops. This work corroborates previous findings that consistent landscape predictors of WNV are difficult to identify across broader geographic areas and sets the stage for additional work that incorporates climate and landscapes interactions for a greater understanding of WNV ecology in this geographic region., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tavares et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Infectious bursal disease virus (IBDV) is an acute and highly infectious RNA virus known for its immunosuppressive capabilities, chiefly inflicting rapid damage to the bursa of Fabricius (BF) of chickens. Current clinical control of IBDV infection relies on vaccination. However, the emergence of novel variant IBDV (nVarIBDV) has posed a threat to the poultry industry across the globe, underscoring the great demand for innovative and effective vaccines. Our previous studies have highlighted the critical role of IBDV VP5 as an apoptosis-inducer in host cells. In this study, we engineered IBDV mutants via a reverse genetic system to introduce amino acid mutations in VP5. We found that the mutant IBDV-VP5/3m strain caused reduced host cell mortality, and that strategic mutations in VP5 reduced IBDV replication early after infection, thereby delaying cell death. Furthermore, inoculation of chickens with IBDV-VP5/3m effectively reduced damage to BF and induced neutralizing antibody production comparable to that of parental IBDV WT strain. Importantly, vaccination with IBDV-VP5/3m protected chickens against challenges with nVarIBDV, an emerging IBDV variant strain in China, reducing nVarIBDV loads in BF while alleviating bursal atrophy and splenomegaly, suggesting that IBDV-VP5/3m might serve as a novel vaccine candidate that could be further developed as an effective vaccine for clinical control of IBD. This study provides a new clue to the development of novel and effective vaccines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: Since 2016, a highly lethal visceral gout induced by infection with the novel goose astrovirus (GoAstV) resulted in an ongoing outbreak in goslings in China, with a mortality rate ranging from 10% to 50%, and causing considerable economic losses in the goose industry. However, the pathogenesis of GoAstV and the molecular mechanism by which kidney lesions are induced by GoAstV infection are unclear., Methods: In the present study, a GEK cell infection model for GoAstV was established, and the apoptosis, inflammatory and innate immune responses induced by GoAstV were investigated in GEK cells., Results: The results shown that the expression of proapoptotic proteins, including Bax, caspase-3, caspase-9 and cytochrome c , increased in the infection group; however, the expression of the antiapoptotic protein Bcl-2 decreased, indicating that apoptosis was induced by GoAstV infection in GEK cells. Besides, the activation of the RIG-I/MDA5 pathway and the downstream upregulation of proinflammatory cytokines, including the adapter proteins MAVS, IRF7 and NF-κB and the proinflammatory cytokines IL-6, IL-8 and TNF-α, were detected in GEK cells infected with GoAstV. In addition, GoAstV infection induces the activation of the NLPR3 pathway and further stimulates the increased production of IL-1β. In summary, the present study revealed that GoAstV infection could induce apoptosis and the activation of the RIG-I/MDA5 and NLRP3 pathways in GEK cells, as well as the massive release of proinflammatory cytokines., Discussion: These results are helpful for elucidating the molecular mechanism of pathological lesions in the kidney in gout goslings infected with GoAstV and the interaction between GoAstV and the innate immune system of the host., Competing Interests: The authors declare the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hou, Jin, Liang, Wang, Jiang, Cao, Sun, Tian, Liu, Xu and Fu.)

Background: Reticuloendotheliosis virus (REV) is an oncogenic immunosuppressive retrovirus that infects different kinds of avian species; posing significant economic losses to the poultry industry worldwide., Methods: In Egypt, there is an unidentified disease associated with the runting-stunting syndrome with neoplasia, suspected to be REV, that has been continuously monitored in several breeder flocks. To diagnose and analyze REV by cell cultures, enzyme-linked immunosorbent assay (ELISA), histopathological investigation, the polymerase chain reaction (PCR) test, and sequencing analysis, 200 blood samples, and 50 tissue specimens were collected. The current study targets the occurrence and genetic characteristics of a viral neoplastic disease, resembling REV infection, circulating in breeder flocks from 2022 to 2023 in the Ismailia, El-Sharqia, and El-Dakahliya governorates., Result: Here, REV was isolated on chicken embryo fibroblast cell culture; exhibiting cell aggregation, rounding, and cell detachments. Collectively, only 70 serum samples were positive for anti-REV antibodies with seroprevalence rates of 35% based on the ELISA test. The histopathological observation demonstrated lymphoreticular tumors in the liver, spleen, and other examined organs. The immunohistochemical staining method confirmed the REV-positive signals in all examined organs (liver, kidney, spleen, bursa, ovaries) except for the heart. The PCR assay of the LTR gene assessed 370 base pairs with only 5 positive samples with a percentage of 16.6%. Three positive samples were further sequenced and submitted to the Genbank under accession numbers (PP763709, PP763710, PP763711). Phylogenetic analysis of the REV-LTR gene showed that our three isolates (Sharquia-1-REV, Ismilia-2-REV, Mansoura-3-REV) are REV subtype III which predominantly circulated in breeders in Egypt. These three isolates are highest similar to American, Chinese, and Taiwanese REV reference strains, and other Egyptian strains with nucleotide identity percentages of 100%, 99%, and 99%; respectively, and on the amino acid identity level were with (99-100%), (98%, 99%), (99%, 100%); respectively., Conclusions: This study established that REV infection was extensively distributed in the breeders and became one of the causes of the clinical outbreaks of tumors, raising awareness of REV as the causative agent of avian oncogenic disease in Egypt., (© 2024. The Author(s).)

Duck circovirus (DuCV) is widely recognized as a prominent virus in China's duck farming industry, known for its ability to cause persistent infections and significant immunosuppression, which can lead to an increased susceptibility to secondary infections, posing a significant threat to the duck industry. Moreover, clinical evidence also indicates the potential vertical transmission of the virus through duck embryos to subsequent generations of ducklings. However, the limited availability of suitable cell lines for in vitro cultivation of DuCV has hindered further investigation into the molecular mechanisms underlying its infection and pathogenicity. In this study, we observed that oral DuCV infection in female breeding ducks can lead to oviduct, ovarian, and follicular infections. Subsequently, the infection can be transmitted to the fertilized eggs, resulting in the emergence of virus-carrying ducklings upon hatching. In contrast, the reproductive organs of male breeding ducks were unaffected by the virus, thus confirming that vertical transmission of DuCV primarily occurs through infection in female breeding ducks. By analyzing transcriptome sequencing data from the oviduct, we focused on claudin-2 , a gene encoding the tight junction protein CLDN2 located on the cell membrane, which showed significantly increased expression in DuCV-infected oviducts of female breeding ducks. Notably, CLDN2 was confirmed to interact with the unique structural protein of DuCV, namely capsid protein (Cap), through a series of experimental approaches including co-immunoprecipitation (co-IP), GST pull-down, immunofluorescence, and adhesion-blocking assays. Furthermore, we demonstrated that the Cap protein binds to the extracellular loop structural domains EL1 and EL2 of CLDN2. Subsequently, by constructing a series of truncated bodies of the CLDN2 promoter region, we identified the transcription factor SP5 for CLDN2. Moreover, we found that DuCV infection triggers the activation of the MAPK-ERK signaling pathway in DEF cells and ducks, leading to an upregulation of SP5 and CLDN2 expression. This process ultimately leads to the transportation of mature CLDN2 to the cell surface, thereby facilitating increased virus adherence to the target organs. In conclusion, we discovered that DuCV utilizes host CLDN2 proteins to enhance adhesion and infection in oviducts and other target organs. Furthermore, we elucidated the signaling pathways involved in the interaction between DuCV Cap proteins and CLDN2, which provides valuable insights into the molecular mechanism underlying DuCV's infection and vertical transmission., Importance: Although duck circovirus (DuCV) poses a widespread infection and a serious hazard to the duck industry, the molecular mechanisms underlying DuCV infection and transmission remain elusive. We initially demonstrated vertical transmission of DuCV through female breeding ducks by simulating natural infection. Furthermore, a differentially expressed membrane protein CLDN2 was identified on the DuCV-infected oviduct of female ducks, and its extracellular loop structural domains EL1 and EL2 were identified as the interaction sites of DuCV Cap proteins. Moreover, the binding of DuCV Cap to CLDN2 triggered the intracellular MAPK-ERK pathway and activated the downstream transcription factor SP5. Importantly, we demonstrated that intracellular Cap also interacts with SP5, leading to upregulation of CLDN2 transcription and facilitating enhanced adherence of DuCV to target tissue, thereby promoting viral infection and transmission. Our study sheds light on the molecular mechanisms underlying vertical transmission of DuCV, highlighting CLDN2 as a promising target for drug development against DuCV infection., Competing Interests: The authors declare no conflict of interest.

Between 2013 and 2018, the novel A/Anhui/1/2013 (AH/13)-lineage H7N9 virus caused at least five waves of outbreaks in humans, totaling 1,567 confirmed human cases in China. Surveillance data indicated a disproportionate distribution of poultry infected with this AH/13-lineage virus, and laboratory experiments demonstrated that this virus can efficiently spread among chickens but not among Pekin ducks. The underlying mechanism of this selective transmission remains unclear. In this study, we demonstrated the absence of Neu5Gc expression in chickens across all respiratory and gastrointestinal tissues. However, Neu5Gc expression varied among different duck species and even within the tissues of the same species. The AH/13-lineage viruses exclusively bind to acetylneuraminic acid (Neu5Ac), in contrast to wild waterbird H7 viruses that bind both Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The level of Neu5Gc expression influences H7 virus replication and facilitates adaptive mutations in these viruses. In summary, our findings highlight the critical role of Neu5Gc in affecting the host range and interspecies transmission dynamics of H7 viruses among avian species.IMPORTANCEMigratory waterfowl, gulls, and shorebirds are natural reservoirs for influenza A viruses (IAVs) that can occasionally spill over to domestic poultry, and ultimately humans. This study showed wild-type H7 IAVs from waterbirds initially bind to glycan receptors terminated with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). However, after enzootic transmission in chickens, the viruses exclusively bind to Neu5Ac. The absence of Neu5Gc expression in gallinaceous poultry, particularly chickens, exerts selective pressure, shaping IAV populations, and promoting the acquisition of adaptive amino acid substitutions in the hemagglutinin protein. This results in the loss of Neu5Gc binding and an increase in virus transmissibility in gallinaceous poultry, particularly chickens. Consequently, the transmission capability of these poultry-adapted H7 IAVs in wild water birds decreases. Timely intervention, such as stamping out, may help reduce virus adaptation to domestic chicken populations and lower the risk of enzootic outbreaks, including those caused by IAVs exhibiting high pathogenicity., Competing Interests: The authors declare no conflict of interest.

Pigeon paramyxovirus serotype 1 (PPMV-1), an antigenic and host variant of avian paramyxovirus Newcastle disease virus (NDV), primarily originating from racing pigeons, has become a global panzootic. Egypt uses both inactivated PPMV-1 and conventional NDV vaccines to protect pigeons from disease and mortality. However, the impact of prevalent strains and the effectiveness of available vaccines in pigeons in Egypt are unclear. This study investigates the virulence of PPMV-1 (Pigeon/Egypt/Sharkia-19/2015/KX580988) and evaluates available paramyxovirus vaccines in protecting pigeons against a PPMV-1 challenge. Ten-day-old specific-pathogen-free (SPF) embryonated chicken eggs infected with this strain exhibited a mean death time (MDT) of 86.4 ± 5.88 h. The intracerebral pathogenicity index (ICPI) in day-old chickens was 0.8, while pigeons experienced an ICPI of 0.96 and an intravenous pathogenicity index (IVPI) of 2.11. These findings classify the strain as virulent and velogenic. Experimental infection of pigeons with this PPMV-1 strain at 10 6 EID 50 /0.1 mL resulted in a 62.5% mortality rate, displaying nervous and enteric distress. The virus caused extensive lesions in visceral organs, with strong immunohistochemistry signals in all examined organs, indicating the systemic spread of the virus concurrent to its neurotropic and viscerotropic tropism. Furthermore, vaccination using an inactivated PPMV-1 and live NDV LaSota vaccine regimen protected 100% of pigeons against mortality, while with a single NDV LaSota vaccine, it was 62.5%. The PPMV alone or combined with NDV LaSota induced protective levels of haemagglutination inhibition (HI) antibody titres and reduced virus shedding from buccal and cloacal cavities. Based on generalised linear gamma model analysis, both PPMV-1 and NDV LaSota are antigenically comparable by HI. These findings suggest that using both inactivated PPMV-1 (G-VI) and live attenuated NDV (LaSota) vaccines is an effective prophylactic regimen for preventing and controlling PPMV-1 and NDV in pigeons, thereby reducing the risk of interspecies transmission.

Avian Leukosis Virus (ALV) is a retrovirus that induces immunosuppression and tumor formation in poultry, posing a significant threat to the poultry industry. Currently, there are no effective vaccines or treatments for ALV. Therefore, the early diagnosis of infected flocks and farm sanitation are crucial for controlling outbreaks of this disease. To address the limitations of traditional diagnostic methods, which require sophisticated equipment and skilled personnel, a dual-tube detection method for ALV-J based on reverse transcription isothermal amplification (RAA) and the CRISPR-Cas13a system has been developed. This method offers the advantages of high sensitivity, specificity, and rapidity; it is capable of detecting virus concentrations as low as 5.4 × 10 0 copies/μL without cross-reactivity with other avian viruses, with a total testing time not exceeding 85 min. The system was applied to 429 clinical samples, resulting in a positivity rate of 15.2% for CRISPR-Cas13a, which was higher than the 14.7% detected by PCR and 14.2% by ELISA, indicating superior detection capability and consistency. Furthermore, the dual-tube RAA-CRISPR detection system provides visually interpretable results, making it suitable for on-site diagnosis in remote farms lacking laboratory facilities. In conclusion, the proposed ALV-J detection method, characterized by its high sensitivity, specificity, and convenience, is expected to be a vital technology for purification efforts against ALV-J., Competing Interests: The authors declare no conflict of interest.

The emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza (HPAI) viruses in 2021 has led to unprecedented epidemics in poultry, changing epidemiological patterns of year-round infections in resident wild avifauna and more frequent spill-over events to mammals. Given this situation, it is important that we recognise that traditional HPAI management strategies are no longer sufficient, and policy changes are required. Poultry vaccination has emerged as a crucial intervention in the current control of HPAI, as evidenced by France's nationwide campaign targeting domestic ducks. However, due to the logistical challenges and potential trade implications of vaccination, broader structural reforms appear also necessary. These include a shift from farm-level to territorial-level biosecurity approaches, putting into practice the concept of 'regional biosecurity'. Given the role duck farm density has played in successive HPAI epidemics in France, there is a need to think about the spatial distribution of poultry farms as a structural component of regional biosecurity and to consider the reduction of farm concentration as a measure to prevent viral spread. The integration of regional biosecurity and poultry vaccination into prevention strategies should impact the way poultry are produced and traded in the future.

BackgroundOutbreaks of highly pathogenic avian influenza (HPAI) on poultry farms and in wild birds worldwide persists despite intensified control measures. It causes unprecedented mortality in bird populations and is increasingly affecting mammalian species. Better understanding of HPAI introduction pathways into farms are needed for targeted disease prevention and control. The relevance of airborne transmission has been suggested but research involving air sampling is limited and unequivocal evidence on transmission routes is lacking.AimWe aimed to investigate whether HPAI virus from wild birds can enter poultry houses through air inlets by characterising host materials through eukaryote DNA sequencing.MethodsWe collected particulate matter samples in and around three HPAI-affected poultry farms which were cleared and decontaminated before sampling. Indoor measurements (n = 61) were taken directly in the airflow entering through air inlets, while outdoor air samples (n = 60) were collected around the poultry house. Positive controls were obtained from a bird rehabilitation shelter. We performed metabarcoding on environmental DNA by deep sequencing 18S rRNA gene amplicons.ResultsWe detected waterbird DNA in air inside all three, and outside of two, poultry farms. Sequences annotated at species level included swans and tufted ducks. Waterbird DNA was present in all indoor and outdoor air samples from the bird shelter.ConclusionAirborne matter derived from contaminated wild birds can potentially introduce HPAI virus to poultry houses through air inlets. The eDNA metabarcoding could assess breaches in biosecurity for HPAI virus and other pathogens potentially transmitted through air via detection of their hosts.

In this study the pathogenicity, infectivity, and transmissibility of H5N1 highly pathogenic avian influenza (HPAI) clade 2.2.1.2 and H5N8 HPAI clade 2.3.4.4b viruses were evaluated in commercial broilers on days 24 and 31. The mortality rate was 100 % in both challenge viruses and in contact birds either on day 24 or day 31 which confirmed the highly pathogenicity of both clades (2.2.1.2/ 2.3.4.4b) in commercial broilers. Both clades (H5N8 clade 2.3.4.4b/ H5N1 clade 2.2.1.2 viruses) were efficiently replicate within and transmitted between commercial broilers. The H5N8-infected birds shed high titer of viruses from oropharynx and cloaca, which associated with the field spread of AIV-H5N8 in commercial broilers. Mean lesion score in both challenged clades showed similar scores, which confirmed the pathogenicity of both clades in commercial broilers' organs (mainly spleen, cerebellum, thymus, Bursa, Lung) which confirm the neurogenic affinity of the virus. In the central nervous system, non-suppurative encephalitis consisting in multifocal areas of necrosis in cerebral hemispheres, intense spongiosis, neuronal chromatolysis and gliosis were commonly observed. In cerebrum, chromatolysis of Purkinje neurons was a common finding. In the lung, interstitial pneumonia consisting of moderate to severe increase of the cellularity (macrophages and lymphoid cells) in air capillaries and focal areas of necrosis associated with intense viral replication was commonly observed. In lymphoid tissues, including spleen, thymus, and bursa of Fabricius, multifocal areas of necrosis/apoptosis of variable intensity in mononuclear cells were present. Particularly, diffuse necrotic areas were present in the spleen. In the liver, we detected focal areas of necrosis with mild distention of hepatic sinusoids. To conclude the AIV either H5N1 or H5N8 have neurological affinity with immune suppression effect based on necrosis and apoptosis of lymphoid tissues., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Marek's disease (MD), an immunosuppression disease induced by Marek's disease virus (MDV), is one of the significant diseases affecting the health and productive performance of poultry. The roles of circular RNAs (circRNAs) in MD development were poorly understood. In this study, we found a circRNA derived from exon 6 of RUNX family transcription factor 2 (RUNX2) gene, named circRUNX2.2, was highly expressed in chicken tumorous spleens (TS) induced by MDV. Through fluorescence in situ hybridization and nuclear-cytoplasmic separation assay, we determined circRUNX2.2 was mainly located in the nucleus. Knockout experiments confirmed that the flanking complementary sequences (RCMs) mediated its circularization. Gain of function assay and dual luciferase reporter gene assay revealed that circRUNX2.2 could promote the expression of RUNX2 via binding with its promoter region. RNA antisense purification assay and mass spectrometry assay showed circRUNX2.2 could recruit proteins such as CHD9 protein. Knocking down CHD9 expression decreased the expression of RUNX2 gene, which confirmed the positive regulation that circRUNX2.2 on RUNX2 expression was probably facilitated via recruiting CHD9 protein. Functional experiments showed that circRUNX2.2 promoted the proliferation of the MD lymphoma-derived chicken cell line, MDCC-MSB1, which confirmed the potential oncogenic role of circRNX2.2 in tumor development. In conclusion, we found that the RUNX2-derived circRUNX2.2 can positively regulate the transcription of the parental gene RUNX2 in a cis-acting manner. The high expression of circRUNX2.2 in MD tumor tissues indicated that it might mediate MD lymphoma progression., Competing Interests: DISCLOSURES The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study analyzes the effect of avian influenza outbreaks on retail price premiums in the US poultry market. We estimate hedonic price models for eggs, chickens, and turkeys, controlling for quality characteristics, unobserved time, and regional factors. To measure the impact of avian influenza outbreaks we use 2 proxies. The first proxy is a measure of the number of new bird infections at the production level. The second proxy measures online search queries related to the outbreak. The results show that, on average, prices increase across product categories, that is, egg, broiler, and turkey markets, during avian influenza outbreaks. Furthermore, we observe price convergence and reduced dispersion within product categories, which is consistent with the economic theory of asymmetric substitutability between conventional and premium products. Our analysis finds that the HPAI outbreak caused a reduction of the price gap between conventional and premium products., Competing Interests: DISCLOSURES The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study assesses different IBV vaccination regimens in broiler chickens using commercially available live attenuated GI-23 (Egyptian-VAR2) and GI-1 (H120) vaccines. Vaccines were administered at 1, 14 days of age, or both. The ciliostasis test, following wild-type VAR2 challenge at 28 days of age, indicated that classic H120+VAR2 at one day old followed by the VAR2 vaccine at 14 days of age provided the highest level of protection (89.58%). Similarly, administering VAR2 at 1 day of age and classic H120 at 14 days of age demonstrated substantial protection (85.42%). Conversely, administering only classic H120 and VAR2 at one day old resulted in the lowest protection level (54.17%). Tracheal virus shedding quantification and assessment of trachea and kidney degenerative changes were significantly lower in vaccinated groups compared to the unvaccinated-challenged group. In conclusion, a carefully planned vaccination regimen based on homologous vaccination offers the most effective clinical protection in broiler chickens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Goose astrovirus (GAstV) has been widespread in China since 2016, causing significant growth inhibition and gout symptoms in goslings and leading to substantial economic losses in the goose industry. To better understand the epidemiological characteristics of GAstV in Guangdong Province, 682 samples were collected from geese with suspected GAstV infection across different regions of Guangdong Province from January 2022 to January 2024. Virus isolation, identification, and genetic evolution analysis were performed. The results showed that all samples were GAstV positive, with 52.64% co-infected with GAstV-1 and GAstV-2, and 42.38% positive for GAstV-2 alone, indicating that GAstV-2 remains the most prevalent subtype. Additionally, three GAstV isolates were identified using molecular detection, immunofluorescence, and transmission electron microscopy on LMH cells or goose embryos. Compared with GDYJ2304 and other reported GAstV-2 strains, the ORF2 region of the GDYJ2210 isolates lacked 3 bases, and the replication ability of GDYJ2210 was significantly higher than that of GDYJ2304. Whole genome sequence alignment and genetic evolution analysis revealed that the GDFS2209 isolate was located in the GAstV-1 branch, with a sequence similarity of 89.70 to 99.00% to GAstV-1 reference strains. The GDYJ2210 and GDYJ2304 isolates were located in the GAstV-2 branch, showing a sequence similarity of 96.80 to 98.90% to GAstV-2 reference strains. These results demonstrated that the GAstV isolates were highly similar to each other despite being prevalent in 5 different regions of Guangdong Province. These findings enhance the understanding of the genetic diversity and evolution of GAstV and may facilitate the development of effective preventive strategies., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Rapid and accurate detection of goose parvovirus (GPV) is crucial for controlling outbreaks and mitigating their economic impact on the poultry industry. This study introduces recombinase polymerase amplification combined with the Pyrococcus furiosus argonaute (RPA-PfAgo) system, a novel diagnostic platform designed to address the limitations of traditional GPV detection methods. Capitalizing on the rapid DNA amplification of RPA and stringent nucleic acid cleavage by the PfAgo protein, the RPA-PfAgo system offers high specificity and sensitivity in detecting GPV. Our optimization efforts included primer and probe configurations, reaction parameters, and guided DNA selection, culminating in a detection threshold of 10 2 GPV DNA copies per microlitre. The specificity of the proposed method was rigorously validated against a spectrum of avian pathogens. Clinical application to lung tissues from GPV-infected geese yielded a detection concordance of 100%, surpassing that of qPCR and PCR in both rapidity and operational simplicity. The RPA-PfAgo system has emerged as a revolutionary diagnostic modality for managing this disease, as it is a promising rapid, economical, and onsite GPV detection method amenable to integration into broad-scale disease surveillance frameworks. Future explorations will extend the applicability of this method to diverse avian diseases and assess its field utility across various epidemiological landscapes., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The fatal gouty disease caused by goose astrovirus genotype 2 (GAstV-2) still seriously endangers the goose industry in China, causing great economic losses. However, research on its infection mechanism has progressed relatively slowly. VP70 is the structural protein of GAstV-2 and is closely related to virus invasion and replication. To better understand the role of VP70 during GAstV-2 infection, we used immunoprecipitation and mass spectrometry to identify host proteins that interact with VP70. Here, we report that cellular vimentin (VIM) is a host binding partner of VP70. Site-directed mutagenesis showed that amino acid residues 399 to 413 of VP70 interacted with VIM. Using reverse genetics, we found that VP70 mutation disrupts the interaction of VP70 with VIM, which is essential for viral replication. Overexpression of VIM significantly promoted GAstV-2 replication, while knockdown of VIM significantly inhibited GAstV-2 replication. Laser confocal microscopy showed that VP70 protein expression induced the rearrangement of VIM, gradually aggregating from the original uniform grid to the side of the nucleus, and aggregated the originally dispersed GAstV-2 RNA in VIM. This rearrangement was associated with increased VIM phosphorylation caused by GAstV-2. Meanwhile, blocking VIM rearrangement with acrylamide substantially inhibited viral replication. These results indicate that VIM interacts with VP70 and positively regulates GAstV-2 replication, and VIM-VP70 interaction and an intact VIM network are needed for GAstV-2 replication. This study provides a theoretical basis and novel perspective for the further characterization of the pathogenic mechanism of GAstV-2-induced gouty disease in goslings., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A suspected outbreak of duck astrovirus (DAstV) disease occurred in a large Muscovy duck farm in Guangdong Province, China, in June 2022, which severely affected the production performance and health of Muscovy ducks. This study aimed to investigate the prevalence of DAstV disease in Southeast China. Herein, we employed semi-nested PCR ethodto screen 5203 swab and liver samples from 11 Muscovy duck farms in 5 provinces of China for the presence of DAstV. Among them, 1356 samples (26.06%, 1356/5203) tested positive for DAstV, out of which 11 DAstV strains were isolated after 10 generations of blind transmission through Leghorn male hepatoma (LMH) cells and performed their whole-genome sequencing. The alignment results showed that the 11 DAstV isolates exhibited relatively low homology (15.4%-75%) with the astrovirus isolates from other species published in GenBank, whereas their homology (nucleotide: 90.4%-99.99%; amino acid: 94%-99.8%) with the DAstV type 1 (DAstV-1) reference strain was higher, indicating considerable homology. The results indicated that DAstV-1 was the main pathogenic factor. Herein, we successfully recreated the clinical symptoms of natural infection in 28-day-old specific-pathogen-free (SPF) ducks using the DAstV-1-GDB-2022 strain. The primary clinical manifestations included liver enlargement, hemorrhaging, and disruptions in liver function. Additionally, we confirmed the cross-species transmission potential of DAstV-1, marking the first occurrence of clinical symptoms of DAstV in 28-day-old SPF chickens. Our findings provide new perspectives on the epidemiology and pathogenicity of DAstV-1 and may help in advancing the development of DAstV vaccines., Competing Interests: DISCLOSURES No conflict of interest exits in the submission of this manuscript. All the authors listed have approved the manuscript that is enclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Infectious bronchitis virus (IBV) strains of genotype GVIII have been emerging in Europe in the last decade, but no biological characterization has been reported so far. This paper reports the extensive genetic and biological characterization of IBV strain D2860 of genotype GVIII which was isolated from a Dutch layer flock that showed a drop in egg production. Whole genome sequencing showed that it has a high similarity (95%) to CK/DE/IB80/2016 (commonly known as IB80). Cross-neutralization tests with antigens and serotype-specific antisera of a panel of different non-GVIII genotypes consistently gave less than 2% antigenic cross-relationship with D2860. Five experiments using specified pathogen-free chickens of 0, 4, 29 and 63 weeks of age showed that D2860 was not able to cause clinical signs, drop in egg production, false layers or renal pathology. There was also a distinct lack of ciliostasis at both 5 and 8 days post-inoculation at any age, despite proof of infection by immunohistochemical (IHC) staining, RT-PCR and serology. IHC showed immunostaining between 5 and 8 days post inoculation in epithelial cells of sinuses and conchae, while only a few birds displayed immunostaining in the trachea. In vitro comparison of replication of D2860 and M41 in chicken embryo kidney cells at 37°C and at 41°C indicated that D2860 might have a degree of temperature sensitivity that might cause it to prefer the colder parts of the respiratory tract.

The H9N2 avian influenza virus (AIV) is spreading worldwide. Presence of H9N2 virus tends to increase the chances of infection with other pathogens which can lead to more serious economic losses. In a previous study, a regulated delayed lysis Salmonella vector was used to deliver a DNA vaccine named pYL233 encoding M1 protein, mosaic HA protein and chicken GM-CSF adjuvant. To further increase its efficiency, chitosan as a natural adjuvant was applied in this study. The purified plasmid pYL233 was coated with chitosan to form a DNA containing nanoparticles (named CS233) by ionic gel method and immunized by intranasal boost immunization in birds primed by oral administration with Salmonella strain. The CS233 DNA nanoparticle has a particle size of about 150 nm, with an encapsulation efficiency of 93.2 ± 0.12 % which protected the DNA plasmid from DNase I digestion and could be stable for a period of time at 37°. After intranasal boost immunization, the CS233 immunized chickens elicited higher antibody response, elevated CD4 + T cells and CD8 + T cells activation and increased T-lymphocyte proliferation, as well as increased productions of IL-4 and IFN-γ. After challenge, chickens immunized with CS233 resulted in the lowest levels of pulmonary virus titer and viral shedding as compared to the other challenge groups. The results showed that the combination of intranasal immunization with chitosan-coated DNA vaccine and oral immunization with regulatory delayed lytic Salmonella strain could enhance the immune response and able to provide protection against H9N2 challenge., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Avian chlamydiosis is a serious avian infection that carries a significant zoonotic danger to the poultry industry. The respiratory co-infections caused by the low pathogenic avian influenza virus H9N2 (LPAIV H9N2) also cause significant financial losses in the poultry industry. The purpose of this study was to examine the pathogenicity of Chlamydophila psittaci, and LPAIV H9N2 individually and in combination in broiler chickens, as well as to determine whether or not aqueous neem (Azadirachta indica) leaf extract is effective against infections caused by these pathogens. Therefore, 120 broiler cobb chicks were equally divided into 4 groups (30 birds each) with triplicates with 10 birds. Broilers in group 1 (G1) were infected with only C. psittaci, broilers in group 2 (G2) were infected with only LPAIV H9N2, broilers in group 3 (G3) were infected with C. psittaci and LPAIV H9N2, and broilers in group 4 (G4) remained not challenged and non-treated with any therapeutic or preventive treatment (negative control). At 21 d postinfection (dpi), birds in G1, G2, and G3 were divided into 3 subgroups of 10 birds each: subgroup (A) remained infected and untreated (positive control), subgroup (B) infected and received oxytetracycline for 5 consecutive d, and subgroup (C) infected and received 8% aqueous neem leaf extract for 5 consecutive d. The multiplication of C. psittaci in birds in G1, in various tissues was evaluated using Giemsa staining and the data showed that multiplication was much higher in the lung, spleen, and liver from 6 h to 21 dpi, but low in the heart from 8 to 21 dpi. During simultaneous co-infection in G3, the birds developed significant clinical symptoms and postmortem lesions (PM). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect viral shedding from oropharyngeal and cloacal swabs between 2 dpi and 8 dpi, with cycle threshold (CT) values ranging from 22 to 24. In contrast, bacterial shedding began 6 h after infection and continued until 21 dpi, with CT values ranging from 23 to 26. Administration of an aqueous neem leaf extract at an 8% concentration (Group C) resulted in a numerical rise in average body weight across all treatment groups in the third and fourth week, as well as a reduction in LPAIV H9N2 and C. psittaci replication in the respiratory and gut of treated birds compared to those treated with oxytetracycline (Group B). Overall, respiratory co-infections pose a considerable risk to the poultry business, which is a big threat. To control C. psittaci and LPAIV H9N2 in broiler chickens, oral supplementation of 8% aqueous neem leaf extract is recommended. This treatment improves the birds' performance, as evidenced by an increase in their average body weight. In addition, the application of 8% aqueous neem leaf extract lowers C. psittaci replication within tissues and diminishes LPAIV H9N2 shedding., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study investigated the genotype classification and pathogenicity of infectious bursal disease virus (IBDV) circulating in vaccinated broiler chicken farms in Egypt. A total of 150 samples were collected from 30 vaccinated commercial broiler chicken farms and pooled into 30 working samples. IBDV was tested using reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the hypervariable region of the viral protein 2 (hvVP2) and the VP1 gene 5' extremity. Both RT-PCR fragments were sequenced from six samples, and then the obtained nucleotide sequences were analyzed. The IBDV genotypes were identified using nucleotide sequences. Five sequences of the six strains examined were classified as genotype A3B2 for the highly virulent segments A and B (vv-A/vv-B IBDV). Interestingly, this study identified and classified a novel segment-reassortant strain as the A1B2 genotype. Specifically, it involved the segment reassortment of classical virulent segment A (cv-A) with vv-B producing cv-A/vv-B reassortant IBDV. Subsequently, we compared the pathogenicity of reassortant (cv-A/vv-B) IBDV and vvIBDV strains identified in this study. Both strains developed typical IBD clinical signs, postmortem lesions, histopathology, immunohistochemistry, and lesion scores, which were more severe in vvIBDV than reassortant IBDV. In conclusion, this is the first report of the genotype classification based on both genome segments (hvVP2 and VP1) with pathogenicity of IBDV circulating in vaccinated broiler chicken farms and this pathogenicity is more severe in vvIBDV strain than a novel reassortant IBDV strain., (© 2024. The Author(s).)