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Background Indonesia is one of the countries that is endemic to avian influenza virus subtype H9N2. This study aims to compare the molecular characteristics of avian influenza virus (AIV) subtype H9N2 from West Java. Methods Specific pathogen-free (SPF) embryonated chicken eggs were used to inoculate samples. RNA extraction and RT-qPCR confirmed the presence of H9 and N2 genes in the samples. RT-PCR was employed to amplify the H9N2-positive sample. Nucleotide sequences were obtained through Sanger sequencing and analyzed using MEGA 7. Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate's similarity to reference isolates from GenBank. Molecular docking analysis was performed on the HA1 protein of the recent isolate and the A/Layer/Indonesia/WestJava-04/2017 isolate, comparing their interactions with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal. Results RT-qPCR confirmed the isolate samples as AIV subtype H9N2. The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate. Phylogenetically, the recent virus remains within the h9.4.2.5 subclade. Notably, at antigenic site II, the recent isolate featured an amino acid N at position 183, unlike A/Layer/Indonesia/WestJava-04/2017. Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal. Conclusion In summary, the recent isolate displayed multiple mutations and a strong affinity for Neu5Ac2-6Gal, commonly found in mammals.

Background Indonesia is one of the countries that is endemic to avian influenza virus subtype H9N2. This study aims to compare the molecular characteristics of avian influenza virus (AIV) subtype H9N2 from West Java. Methods Specific pathogen-free (SPF) embryonated chicken eggs were used to inoculate samples. RNA extraction and RT-qPCR confirmed the presence of H9 and N2 genes in the samples. RT-PCR was employed to amplify the H9N2-positive sample. Nucleotide sequences were obtained through Sanger sequencing and analyzed using MEGA 7. Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate's similarity to reference isolates from GenBank. Molecular docking analysis was performed on the HA1 protein of the recent isolate and the A/Layer/Indonesia/WestJava-04/2017 isolate, comparing their interactions with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal. Results RT-qPCR confirmed the isolate samples as AIV subtype H9N2. The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate. Phylogenetically, the recent virus remains within the h9.4.2.5 subclade. Notably, at antigenic site II, the recent isolate featured an amino acid N at position 183, unlike A/Layer/Indonesia/WestJava-04/2017. Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal. Conclusion In summary, the recent isolate displayed multiple mutations and a strong affinity for Neu5Ac2-6Gal, commonly found in mammals.

Background Indonesia is one of the countries that is endemic to avian influenza virus subtype H9N2. This study aims to compare the molecular characteristics of avian influenza virus (AIV) subtype H9N2 from West Java. Methods Specific pathogen-free (SPF) embryonated chicken eggs were used to inoculate samples. RNA extraction and RT-qPCR confirmed the presence of H9 and N2 genes in the samples. RT-PCR was employed to amplify the H9N2-positive sample. Nucleotide sequences were obtained through Sanger sequencing and analyzed using MEGA 7. Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate's similarity to reference isolates from GenBank. Molecular docking analysis was performed on the HA1 protein of the recent isolate and the A/Layer/Indonesia/WestJava-04/2017 isolate, comparing their interactions with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal. Results RT-qPCR confirmed the isolate samples as AIV subtype H9N2. The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate. Phylogenetically, the recent virus remains within the h9.4.2.5 subclade. Notably, at antigenic site II, the recent isolate featured an amino acid N at position 183, unlike A/Layer/Indonesia/WestJava-04/2017. Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal. Conclusion In summary, the recent isolate displayed multiple mutations and a strong affinity for Neu5Ac2-6Gal, commonly found in mammals.

Study on sialidases as antiviral agents has been widely performed, but many types of sialidase have not been tested for their antiviral activity. Pasteurella multocida NanB sialidase is one such sialidase that has never been isolated for further research. In this study, the activity of NanB sialidase was investigated in silico by docking the NanB sialidase of Pasteurella multocida to the Neu5Acα(2–6)Gal and Neu5Acα(2–3)Gal ligands. Additionally, some local isolates of Pasteurella multocida , which had the NanB gene were screened, and the proteins were isolated for further testing regarding their activity in hydrolyzing Neu5Acα(2–6)Gal and Neu5Acα(2–3)Gal. Silico studies showed that the NanB sialidase possesses an exceptional affinity towards forming a protein–ligand complex with Neu5Acα(2–6)Gal and Neu5Acα(2–3)Gal. NanB sialidase of Pasteurella multocida B018 at 0.129 U/mL and 0.258 U/mL doses can hydrolyze Neu5Acα(2–6)Gal and Neu5Acα(2–3)Gal better than other doses. In addition, those doses can inhibit effectively H9N2 viral binding to red blood cells. This study suggested that the NanB sialidase of Pasteurella multocida B018 has a potent antiviral activity because can hydrolyze sialic acid on red blood cells surface and inhibit the H9N2 viral binding to the cells.

Background and Aim: Fowl avidenoviruses (FAdVs) are generally considered ubiquitous, but certain serotypes and strains are known to be associated with primary diseases, such as inclusion body hepatitis (IBH). Since 2018, the outbreak of IBH has been reported in part provinces of Indonesia. This study aimed to isolate and molecularly characterize the FAdV from Banten and West Java Provinces of Indonesia and described the phylogenetic relationship with the FAdV that has been characterized in other countries. Materials and Methods: A total of 25 FAdV archive samples have been collected from January to August 2019 from clinical cases of FAdV infection in Banten and West Java Provinces, Indonesia. Collected samples were inoculated in 10-day-old specific-pathogenic-free chicken embryonated eggs. Hexon gene of FAdV was detected using polymerase chain reaction (PCR) with a primer set from previous study. To gain a better understanding of the FAdV genetic properties and construct the phylogeny tree, the PCR products were sequenced and subjected to a BLAST search and inferred using the neighbor-joining method by bootstrap test 1000×. Results: FAdV-D and FAdV-E are present in Banten, Indonesia. The phylogenetic analysis of 850 nucleotides that encode 289 amino acid of the partial hexon gene shows that the isolates Broiler/MSL/Ciputat-149/18, Broiler/MSL/Lebak-151/18, and Broiler/MSL/Ciputat-29/19 have 100% homology with FAdV-E TR/BVKE/R/D-1 from Turkey, whereas the isolates Layer/MSL/Ciputat-20/19 and Broiler/MSL/Ciputat-30/19 have 100% homology with FAdV-D strain 685 from Canada. Conclusion: The present study provides updates of the circulating FAdV in commercial poultry flocks in Banten and West Java Provinces, Indonesia. Since the FAdV vaccine was unavailable in Indonesia, this result might be used as guidance to select a proper FAdV vaccine strain. Our result indicates that at least two FAdV species were circulating among poultry in Banten and West Java Provinces, Indonesia; they are FAdV-D and FAdV-E.

Sialidases, enzymes produced by Clostridium perfringens Type A, play a critical role in cleaving sialic acid residues essential for viral entry into host cells. By targeting pathogens such as coronaviruses, influenza, and paramyxoviruses, sialidase represents a promising therapeutic candidate. While in vitro studies confirm its efficacy against influenza, evaluating its safety profile in vivo is imperative. This study investigates the acute and subacute toxicity of sialidase from C. perfringens Type A in BALB/c mice ( Mus musculus ). Acute toxicity involved a single intranasal dose followed by a 14‐day observation, while subacute toxicity encompassed daily doses for 30 days. Mice were administered 187.5, 375, or 750 mU/mL of sialidase, with saline as the control. No mortality or overt toxicity occurred, but significant histopathological alterations were evident in the lungs and liver at higher doses. Observed effects included lung inflammation and edema, liver congestion, and kidney inflammation. Hematological analysis revealed immunosuppressive effects, including reduced white blood cell and lymphocyte counts, alongside dose‐dependent IL‐6 expression changes. Sialidase doses of 187.5 and 375 mU/mL were deemed safe, whereas toxicity became pronounced at 750 mU/mL.

The avian influenza virus (AIV) subtype H9N2 circulating in Indonesia has raised increasing concern about its impact on poultry and its public health risks. In this study, the H9N2 virus from chicken poultry farms in Java was isolated and characterized molecularly. Thirty-three pooled samples of chicken brain, cloacal swab, trachea, and oviduct were taken from multiple chickens infected with AIV in five regions of Java, Indonesia. The samples were isolated from specific pathogenic-free embryonated eggs that were 9 days old. Reverse transcription polymerase chain reaction and sequencing were used to identify H9N2 viruses. This study was successful in detecting and characterizing 13 H9N2 isolates. The sequencing analysis of genes revealed a 96.9%-98.8% similarity to the H9N2 AIV isolated from Vietnam in 2014 (A/muscovy duck/Vietnam/LBM719/2014). According to the phylogenetic analysis, all recent H9N2 viruses were members of the lineage Y280 and clade h9.4.2.5. Nine of the H9N2 isolates studied showed PSKSSR↓GLF motifs at the cleavage site, while four had PSKSSR↓GLF. Notably, all contemporary viruses have leucine (L) at position 216 in the receptor-binding region, indicating that the virus can interact with a human-like receptor. This study described the features of recent H9N2 viruses spreading in Java's poultry industry. Additionally, H9N2 infection prevention and management must be implemented to avoid the occurrence of virus mutations in the Indonesian poultry industry.

Objective: This study aims to evaluate the effect of Clostridium perfringens sialidase treatment on monolayer cell behavior using computational screening and an in vitro approach to demonstrate interaction between enzyme-based drugs and ligands in host cells. Materials and Methods: The in silico study was carried out by molecular docking analysis used to predict the interactions between atoms that occur, followed by genetic characterization of sialidase from a wild isolate. Sialidase, which has undergone further production and purification processes exposed to chicken embryonic fibroblast cell culture, and observations-based structural morphology of cells compared between treated cells and normal cells without treatment. Results: Based on an in silico study, C. perfringens sialidase has an excellent binding affinity with Neu5Acα (2.3) Gal ligand receptor with Gibbs energy value (ΔG)—7.35 kcal/mol and Ki value of 4.11 μM. Wild C. perfringens isolates in this study have 99.1%–100% similarity to the plc gene, NanH, and NanI genes, while NanJ shows 93.18% similarity compared to the reference isolate from GenBank. Sialidase at 750 and 150 mU may impact the viability, cell count, and cell behavior structure of fibroblast cells by significantly increasing the empty area and perimeter of chicken embryo fibroblast (CEF) cells, while at 30 mU sialidase shows no significant difference compared with mock control. Conclusion: Sialidase-derived C. perfringens has the capacity to compete with viral molecules for attachment to host sialic acid based on in silico analysis. However, sialidase treatment has an impact on monolayer cell fibroblasts given exposure to high doses.

Objective: The Newcastle disease virus (NDV) is an infectious disease that causes very high eco¬nomic losses due to decreased livestock production and poultry deaths. The vaccine’s ineffec¬tiveness due to mutation of the genetic structure of the virus impacts obstacles in controlling the disease, especially in some endemic areas. This study aimed to provide an alternative treatment for NDV infection by observing the viral replication inhibitor activity of Clostridium perfringens sialidase in primary chicken embryo fibroblast (CEF) cells. Materials and Methods: The virus was adapted in CEF monolayer cells, then collected thrice using the freeze–thaw method and stored at −20°C for the next step in the challenge procedure. C. perfringens crude sialidase was obtained, but it was further purified via stepwise elution in ion exchange using Q Sepharose® Fast Flow and affinity chromatography with oxamic acid agarose. The purified sialidase was tested for its toxicity, ability to breakdown sialic acid, stopping viral replication, and how treated cells expressed their genes. Results: According to this study, purified C. perfringens sialidase at dosages of 187.5, 93.75, and 46.87 mU effectively hydrolyzes CEF cells’ sialic acid and significantly inhibits viral replication on the treated cells. However, sialidase dosages of 375 and 750 mU affected the viability of mono¬layer CEF cells. Interestingly, downregulation of toll-like receptor (TLR)3 and TLR7 (p < 0.05) in the sialidase-treated group indicates viral endocytosis failure. Conclusions: By stopping endocytosis and viral replication in host cells, sialidase from C. perfrin¬gens can be used as an alternative preventive treatment for NDV infection.