Avian Influenza Virus Subtype H5N1 Research Articles

Semi-Scavenging Poultry as Carriers of Avian Influenza Genes.

Ducks are the natural reservoir of influenza A virus and the central host for the avian influenza virus (AIV) subtype H5N1, which is highly pathogenic. Semi-scavenging domestic ducks allow for the reemergence of new influenza subtypes which could be transmitted to humans. We collected 844 cloacal swabs from semi-scavenging ducks inhabiting seven migratory bird sanctuaries of Bangladesh for the molecular detection of avian influenza genes. We detected the matrix gene (M gene) using real-time RT–PCR (RT–qPCR). Subtyping of the AIV-positive samples was performed by RT–qPCR specific for H5, H7, and H9 genes. Out of 844 samples, 21 (2.488%) were positive for AIV. Subtyping of AIV positive samples (n = 21) revealed that nine samples (42.85%) were positive for the H9 subtype, five (23.80%) were positive for H5, and seven (33.33%) were negative for the three genes (H5, H7, and H9). We detected the same genes after propagating the virus in embryonated chicken eggs from positive samples. Semi-scavenging ducks could act as carriers of pathogenic AIV, including the less pathogenic H9 subtype. This can enhance the pathogenicity of the virus in ducks by reassortment. The large dataset presented in our study from seven areas should trigger further studies on AIV prevalence and ecology.

Characterization of a monoclonal antibody against the hemagglutinin stem of H7N9 subtype avian influenza virus

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (431W-433Y-437L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.

Seroprevalence of Avian Influenza virus subtype H5N1 in chickens of the two main poultry market in Surakarta area, Central Java

Avian influenza (AI) subtype H5N1 virus is very contagious among domestic poultry such as chicken (Gallus gallus) and potentially infecting humans through direct or indirect contact with the infected animal. In the Surakarta area, Central Java, there are two major poultry markets, Silir poultry market and Jumantono farm. Here, we determined the seroprevalence of the AI virus in chickens sold in both poultry markets. The method was conducted using an explorative experiment by collecting 140 samples of chicken blood serum. The presence of antibodies to the AI virus subtype H5N1 was measured through a standard serological test via hemagglutination-inhibition (HI) assay. The HI assay detected a large antibody difference against AI from samples of both locations; 4.28% from Silir poultry market and 48.57% from Jumantono farm. Because the chickens from the Silir poultry market were not vaccinated, the level of antibody detected indicatesd that some chickens were exposed to the virus through infection. Meanwhile, the high positivity rate in Jumantono farm might reflect the presence of neutralizing antibody, since the chickens from this farm received periodic vaccinations. Continuous quality surveillance in the environment of the poultry market is essential to reduce the impact of AI outbreaks against chickens and humans.

Antigen-capture ELISA and immunochromatographic test strip to detect the H9N2 subtype avian influenza virus rapidly based on monoclonal antibodies

BackgroundThe H9N2 subtype of avian influenza virus (AIV) has become the most widespread subtype of AIV among birds in Asia, which threatens the poultry industry and human health. Therefore, it is important to establish methods for the rapid diagnosis and continuous surveillance of H9N2 subtype AIV.MethodsIn this study, an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a colloidal gold immunochromatographic test (ICT) strip using monoclonal antibodies (MAbs) 3G4 and 2G7 were established to detect H9N2 subtype AIV.ResultsThe AC-ELISA method and ICT strip can detect H9N2 subtype AIV quickly, and do not cross-react with other subtype AIVs or other viruses. The detection limit of AC-ELISA was a hemagglutinin (HA) titer of 4 for H9N2 subtype AIV per 100 μl sample, and the limit of detection of the HA protein of AIV H9N2 was 31.5 ng/ml. The ICT strip detection limit was an HA titer of 4 for H9N2 subtype AIV per 100 μl sample. Moreover, both detection methods exhibited good reproducibility and repeatability, with coefficients of variation < 5%. For detection in 200 actual poultry samples, the sensitivities and specificities of AC-ELISA were determined as 93.2% and 98.1%, respectively. The sensitivities and specificities of the ICT strips were determined as 90.9% and 97.4%, respectively.ConclusionsThe developed AC-ELISA and ICT strips displayed high specificity, sensitivity, and stability, making them suitable for rapid diagnosis and field investigation of H9N2 subtype AIV.

Molecular characterization and antigenic analysis of reassortant H9N2 subtype avian influenza viruses in Eastern China in 2016

H9N2 avian influenza viruses (AIVs) can cause respiratory symptoms and decrease the egg production. Additionally, H9N2 AIVs can provide internal genes for reassortment with other subtypes. During the monitoring of live poultry markets in 2016, a total of 32 (32/179, 17.88%) H9N2 AIVs were isolated from poultry in Eastern China, and seven representative strains were selected based on the isolation time, isolation location and sequence homology for further characterization. Phylogenetic analysis of hemagglutinin and neuraminidase showed that these H9N2 AIVs clustered into the Y280 sublineage. And the phylogenetic trees of six internal genes showed that the source of these gene fragments was more abundant, suggesting that extensive reassortment has occurred in these H9N2 viruses. Molecular analysis showed that multiple specific amino acid mutations occurred that increased H9N2 AIVs’ infectivity, transmissibility, and affinity to mammals, including Q226L and Q227M in hemagglutinin, E627K in polymerase basic protein 2 (PB2), L13P in polymerase basic protein 1 (PB1), and A70V and S409N in polymerase acidic protein (PA). Pathogenicity tests in mice showed these H9N2 AIVs could replicate in lungs and exhibited slight to moderate virulence. The continuous circulation of these H9N2 viruses suggests the necessity for persistent surveillance of the H9N2 AIVs in poultry.

Recombinant HA1-ΔfliC enhances adherence to respiratory epithelial cells and promotes the superiorly protective immune responses against H9N2 influenza virus in chickens

H9N2 subtype avian influenza virus (AIV) is an ongoing threat causing substantial loss to the poultry industry and thus necessitating the development of safe and effective vaccines against AIV. Given that inactivated vaccines are less effective in activating the mucosal immune system, we aimed to generate a vaccine that can actively engage the mucosal immunity which is the front line of the immune system. We generated a group of flagellin-based hemagglutinin globular head (HA1) fusion proteins and characterized their immunogenicity and efficacy. We found that Salmonella typhimurium flagellin (fliC) lacking the hypervariable domain (called herein as HA1-ΔfliC) was recognized by TLR5 and induced a moderate innate immune response compared to N-terminus of fliC (HA1-fliC) and C-terminus of fliC (fliC-HA1). The HA1-ΔfliC protein had increased adherence to the nasal cavity and trachea than HA1-fliC and fliC-HA1 and significantly increased the HA-specific sIgA titers. Our in vivo results revealed that chickens treated with HA1-ΔfliC had a significantly reduced level of viral loads in the cloaca and throat compared with chickens treated with inactivated vaccine. Overall, these results revealed that HA1-ΔfliC can protect chickens against H9N2 AIV by eliciting the efficient mucosal immune responses.

G1-like PB2 gene improves virus replication and competitive advantage of H9N2 virus.

H9N2 subtype avian influenza virus has dramatically evolved and undergone extensive reassortment since its emergence in early 1990s in China. The genotype S (G57), emerging in 2007 with the substitution of F98-like PB2 and M gene by G1-like ones, has become the overwhelming predominant genotype for the past 11years since 2010. Here, we found that virus with G1-like PB2 were more efficient in protein expression and in infectious virus production than that with F98-like PB2 gene. By coinfected MDCK cells with the reassortant virus, more survival opportunity for viruses with G1-like PB2 than that of F/98-like was observed. Besides, in animal experiments, we found that the G1-like PB2 increases virus infectivity, replication, and virus shedding of H9N2 in chickens. Our results suggested that the substitution of G1-like PB2 play important role in promoting the fitness of genotype S H9N2 virus in China.

Seroprevalence and associated risk factors of avian influenza virus subtype H9N2 in backyard poultry of Peshawar Pakistan

The current study was conducted to estimate the serological prevalence and risk factors associated with the transmission of avian influenza subtype H9N2 among backyard poultry from different villages of semi-arid climate district Peshawar Pakistan between January to May 2019. In total, 240 blood samples of healthy backyard poultry older than two months were collected from 30 different villages of district Peshawar. A predesigned questionnaire was used to collect the data related to risk factors. Se-rum samples were tested through the haemagglutination inhibition (HI) test further confirmed by the micro-neutralizing test (MN). Antibody body titer ≤ 8 considered negative for prevalence of H9N2. In order to classify risk factors, Chi-square and Logistic regression analyses were performed. Out of 240, 150 were found to be positive. The overall seroprevalence was 62.5%, mean antibody titer for avian influenza virus in all villages was 6.8 and (95%) confidence interval ranges from 35.33 to 51.70% re-spectively. The high prevalence of antibodies to avian influenza viruses in bird serum emphasizes that avian influenza plays an important role in the respiratory infections of backyard chickens in the area, and possibly throughout Pakistan. In order to avoid the introduction of H9N2, biosecurity initiatives, surveillance, monitoring systems, and to some extent, vaccinations are useful tools.

Pathogenesis of Avian Influenza Virus Subtype H9N2 in Turkeys and Evaluation of Inactivated Vaccine Efficacy.

Avian influenza H9N2 viruses circulate in all types of poultry species, including turkeys, and cause significant losses for the poultry industry in many parts of the word. The aim of this study was to assess the pathogenesis of the Moroccan avian influenza virus (AIV) H9N2 under experimental conditions in turkeys and the protection efficacy of an inactivated commercial vaccine against AIV H9N2. Unvaccinated turkeys showed marked depression sinusitis, respiratory distress characterized by bronchiolar and tracheal rales of moderate severity, and a mortality rate of 50%. Postmortem examinations of dead and euthanatized birds revealed the presence of fibrinous tracheitis and airsacculitis lesions. Vaccination reduced the mortality rate to 20%. Vaccinated birds recovered at day 10 postchallenge, and only 12.5% (1/8) and 37.5% of birds still displayed fibrinous and nonfibrinous airsacculitis lesions, respectively, at day 15 postinoculation. Viral shedding in cloacal and tracheal swabs was lower in vaccinated than in control birds. Although viral RNA was detected in the cloacal swabs of all unvaccinated turkeys at day 3 postinoculation, only 50% of the vaccinated turkeys were positive for virus detection. At day 11 postinoculation, no viral RNA was detected in oropharyngeal swabs of vaccinated turkeys, whereas 40% of the unvaccinated turkeys were still shedding virus.

Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 104 egg infective dose 50 (EID50) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID50 105), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.

The effect of garlic (Allium sativum) and red onion (Allium cepa) on the histopathological signs of avian influenza disease in broiler chickens exposed to avian influenza virus subtype H9N2

ویروس آنفلوانزای پرندگان، تحت­تیپ H9N2در ایران شیوع بالائی داشته و از سراسر جهان هم گزارش شده­ است. وقوع بیماری آنفلوانزا باعث خسارت سنگین اقتصادی در صنعت طیور می‌شود. در مطالعه حاضر نقش سیر (Allium sativum) و پیاز قرمز (Allium cepa) در کاهش شدت نشانه­های آسیب‌شناختی بیماری آنفلوانزای پرندگان ناشی از تحت­تیپ H9N2 در جوجه­های گوشتی، بررسی شد. بدین منظور ابتدا سمیت سیر و پیاز مورد بررسی قرار­گرفت. همچنین تعداد 72 قطعه جوجه­ء یک روزه نژاد گوشتی سویه­­ء راس به 9 گروه 8 قطعه­ای تقسیم ­شدند که شامل‌ 4 گروه درمانی، 1 گروه کنترل مثبت و 4 گروه مسمومیت بودند. سپس گروه‌های درمانی در 28 روزگی با ویروس مواجهه داده شدند. پرندگان گروه‌های مواجهه داده شده یک روز بعد از مواجهه به مدت هفت روز، به‌طور جداگانه سیر و پیاز قرمز را همراه غذا دریافت کردند. در ادامه علائم بالینی، نشانه­های آسیب‌شناختی و نیز تیتر آنتی­بادی اختصاصی ضد ویروس آنفلوانزای تحت­تیپ H9N2 در سرم پرندگان مورد آزمایش بررسی شد. سیر و پیاز هیچ اثر سمی بر جوجه‌ها نداشتند. در آسیب‌شناسی روده نیز میزان افزایش سلول‌های جامی‌شکل و قطر پرزها در گروه‌های کنترل منفی و درمانی در مقایسه با گروه کنترل مثبت، بیشتر بود که از نظر آماری نیز اختلاف معنی‌دار داشتند (05/0p<). به نظر می­رسد سیر و پیاز قرمز که موجب کاهش شدت علائم آسیب‌شناختی بیماری آنفلوانزای پرندگان ناشی از تحت­تیپ H9N2 ویروس آنفلوانزا در دئودنوم جوجه­های گوشتی مورد مطالعه شدند، احتمالا با کاهش تعداد ویروس‌ها، در ترمیم مخاط روده نقش دارند.

NF-кB pathway genes expression in chicken erythrocytes infected with avian influenza virus subtype H9N2

ABSTRACT 1. Chicken erythrocytes in blood vessels are the most abundant circulating cells, which participate in the host’s immune responses. The transcription factor nuclear factor-kappa B (NF-κB) plays a vital role in the inflammatory response following viral infections. However, the expression of the NF-κB pathway, and other immune-related genes in chicken erythrocytes infected with low pathogenic avian influenza virus (LPAIV H9N2), has not been extensively studied. 2. The following study determined the interaction of LPAIV H9N2 with chicken erythrocytes using indirect immunofluorescence microscopy. This was followed by investigating myeloid differentiation primary response 88 (MyD88), C-C motif chemokine ligand 5 (CCL5), melanoma differentiation-associated protein 5 (MDA5), the inhibitor of nuclear factor-kappa B kinase subunit epsilon (IKBKE), NF-κB inhibitor alpha (NFKBIA), NF-κB inhibitor epsilon (NFKBIE), interferon-alpha (IFN-α), colony-stimulating factor 3 (CSF3) and tumour necrosis factor receptor-associated factor 6 (TRAF6) by mRNA expression using quantitative real-time PCR (qRT-PCR) at four different time intervals (0, 2, 6 and 10 h). 3. There was a significant interaction between erythrocytes and LPAIV H9N2 virus. Furthermore, the mRNA expression of the NF-κB pathway and other immune-related genes were significantly up-regulated at 2 h post-infection in infected chicken erythrocytes, except for TRAF6, which were significantly downregulated. While at 0 h post-infection, IFN-α and CSF3 were significantly upregulated, whereas NFKBIA was significantly downregulated. Further expression of MDA5, CCL5 and NFKBIA was upregulated, while TRAF6 was downregulated at 6 h post-infection. In infected erythrocytes, expression of MyD88, CCL5 and IKBKE was upregulated. However, IFN-α and TRAF6 were downregulated at 10 h post-infection. 4. These results give initial evidence that the NF-κB pathway, and other genes related to immunity, in chicken erythrocytes may contribute to LPAIV subtype H9N2 and induce host immune responses.

Molecular Characterizations of a High Pathogenic Avian Influenza H5N8 in Iraq

High pathogenicity avian influenza subtype H5N8 viruses were detected in different areas in Iraq at thelast 2017 and early 2018 and 2019 and the disease was detected clinically. This disease is responsible forhigh economic losses for poultry industry and threat human health, so that, this study was conducted formolecular detection, characterization and phylogenic analysis of avian influenza in Iraq. AI subtype H5N8 isan infectious disease primarily in birds and responsible for severe respiratory illness which associated witha high percentage of morbidity and mortality in wild and domestic birds. During this study one hundred fiftydifferent samples including (trachea, larynx and lung) were collected from different areas of broiler chickenfrom Baghdad and its surrounding regions during winter 2019. Avian influenza virus subtype H5N8 wasdetected by using real time RT-PCR technique, and specific kits (KyltR Germany) for AIV subtypes H5 andN8 were used respectively. The results revealed that (15) samples out of (150) collected samples were gavepositive results for avian influenza H5 these positive samples were prepared for a second step of detectionby RT-PCR test specific for N8 subtype the results revealed that only (12) out of (15) tested samples werepositive for H5N8 (8%).Genetic sequencing of isolates and phylogenic analysis of three selected isolates of(H5N8) bellowing to different areas indicate that all strains bellowing to H5 class (2.3.4.4) high pathogenicavian influenza revealed that they are closely related to Egyptian strain (A/ duck/ Egypt/ F446/2017.H5N8-MH893737.1) (with 97.6% identity). Analysis of the mono-basic amino acid (PQIEPR / GLF) atthe hemagglutinin cleavage site revealed there is no deletion of the stalk region with the neuraminidaseindicated that the isolates is a typical HPAI strain (A / duck / Egypt / F446/2017). The similarity of thenucleotide sequence analysis of hemagglutinin gene revealed that there was a high homology (97.6%) to thatof A/duck/Egypt/F446/2017 H5N8 .

Molecular characteristics of the H9N2 avian influenza viruses in live poultry markets in Anhui Province, China, 2013 to 2018.

Background and AimsH9N2 subtype avian influenza virus (AIV) has low‐pathogenicity but causes respiratory symptoms and drop in egg production in chicken with long‐term virus shedding, resulting in great economic losses due to high mortality related to secondary infection with other pathogens. In recent years, H9N2 viruses have been posing a threat to public health, causing human infection in China. Compared to studies on other AIV subtypes, there are relatively few studies on the pathogenic mechanism of the H9N2 virus in mammals. H9N2 subtype AIV has been circulating worldwide in many avian species and transmitting with high efficiency in poultry. It can provide internal genes for other subtypes to produce new viruses, causing a pandemic risk. It is important to carry out long‐term surveillance and pathogenic characteristics of the H9N2 virus. In this study, we conducted environmental surveillance of live poultry markets in Anhui province from 2013 to 2018, and 33 representative environmental isolates were selected and studied systematically.MethodsThe genomic RNA of Anhui H9N2 isolates was subjected to RT‐PCR amplification followed by sequencing analysis.ResultsThirty‐three strains were isolated from the embryonated eggs of specific‐pathogen‐free chickens. Phylogenetic analysis indicated that h9.4.2.5‐like H9N2 viruses were predominant during 2013‐2018 and acquired multiple specific amino acid mutations that may have increased their affinity for mammals and enhanced their infectivity and transmissibility. Additionally, six internal genes of H9N2 clustered together with the novel human‐lethal reassortant viruses, such as the low‐pathogenicity H7N9, H10N8, and Anhui H5N6 viruses, and even HPAI H7N9.ConclusionBecause H9N2 viruses may be the donors of internal genes that lead to the generation of novel reassortant viruses with enhanced pathogenicity in Anhui province, continuous environmental surveillance of live poultry markets, a key source of reassorted H9N2 and other avian influenza viruses, is of great importance.

Molecular epidemiology and pathogenicity of H5N1 and H9N2 avian influenza viruses in clinically affected chickens on farms in Bangladesh

ABSTRACT Avian influenza virus (AIV) subtypes H5N1 and H9N2 co-circulate in poultry in Bangladesh, causing significant bird morbidity and mortality. Despite their importance to the poultry value chain, the role of farms in spreading and maintaining AIV infections remains poorly understood in most disease-endemic settings. To address this crucial gap, we conducted a cross-sectional study between 2017 and 2019 in the Chattogram Division of Bangladesh in clinically affected and dead chickens in farms with suspected AIV infection. Viral prevalence of each subtype was approximately 10% among farms for which veterinary advice was sought, indicating high levels of virus circulation in chicken farms despite the low number of reported outbreaks. Co-circulation of both subtypes was common in farms, with our findings suggest that in the field, the co-circulation of H5N1 and H9N2 can modulate disease severity, which could facilitate an underestimated level of AIV transmission in the poultry value chain. Finally, using newly generated whole-genome sequences, we investigate the evolutionary history of a small subset of H5N1 and H9N2 viruses. Our analyses revealed that for both subtypes, the sampled viruses were genetically most closely related to other viruses isolated in Bangladesh and represented multiple independent incursions. However, due to lack of longitudinal surveillance in this region, it is difficult to ascertain whether these viruses emerged from endemic strains circulating in Bangladesh or from neighbouring countries. We also show that amino acids at putative antigenic residues underwent a distinct replacement during 2012 which coincides with the use of H5N1 vaccines.

Molecular Characterization, Pathogenesis and Immunological Effect of Avian Influenza H9N2 Virus Isolated From Broiler Chickens in Egypt During 2019

Many commercial flocks in Egypt were infected with the low pathogenic avian influenza virus (LPAIV) subtype H9N2, resulting in severe economic losses, particularly when combined with other viral and bacterial diseases. The current study aimed to investigate the genetic evolution of LPAIV (H9N2) in broiler farms in Egypt during 2019 and evaluate its effect on the viral pathogenesis, cytokine response, innate immunity and oxidative state in specific pathogen free (SPF) chicks. Sixty tracheal swabs were collected from broilers and tested by RRT-PCR and the HA gene were sequenced for the genetic evolution. Then this isolated virus was experimentally studied in a total of 20 SPF chicks (21 days old) that were divided into two equal groups. Group-1(G1) was negative control and group-2 (G2) was experimentally infected with the isolated LPAIV (H9N2). The obtained results revealed that all isolated LPAIV (H9N2) in this study were belong to G1-like Eurasian sub-lineages (A/quail/Hong Kong/G1/97-like) and clustered with Israeli strains in group B with specific mutation specific for Egyptian AIV-H9N2 isolated in 2018-2019 forming new subgroup-I. The experimental infection with LPAIV (H9N2) (G2) in SPF chickens, induced mild clinical signs, moderate histopathological changes in lung and spleen, shedding of higher mean LPAIV (H9N2) titre in trachea, up regulation of both the pro-inflammatory (IL-6) and the anti-viral (IFN-γ) cytokines, significant decrease of the serum levels of lysozyme and disturbance of the oxidative state where the serum nitric oxide (NO) and the malondialdehyde (MDA) levels were significantly increased and the antioxidants glutathione (GSH) and superoxide dismutase (SOD) were significantly decreased. In conclusion, the LPAIV (H9N2) was continually circulated in broiler chicken farms and highly evolved. The induced oxidative stress in LPAIV (H9N2) infection could be contributed to the viral pathogenesis, up-regulation of pro-inflammatory and anti-viral cytokines and down-regulation of the innate immune response.

Isolation and molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses from poultry in Java, Indonesia.

Objective: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.Materials and Methods: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.Results:This study was successful in detecting and characterizing 13 H9N2 isolates. The sequencing analysis of hemagglutinin 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.Conclusion: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.

Karaterisasi Virus Avian Influenza Subtipe H5N1 Isolat Lapang Asal Bali Untuk Kandidat Vaksin

A research on the isolation and characterization of the Avian Influenza H5N1 subtype field isolate has been carried out at the BSL-3 Laboratory of PT Sanbio Laboratories, Bogor. The aim of the study was to prepare a candidate for the H5N1 subtype Avian Influenza virus vaccine. Virus isolates were taken from field isolates from Bali. A total of seven field H5N1 AI subtypes from Bali were characterized in Bogor. The isolates were: isolate 3A, isolate 4A, isolate 9C, isolate 10 A, isolate 10 C, isolate P65, isolate P67. The passage of isolates was carried out on 9-day-old embryonic Specific Pathogenic (SPF) chicken eggs by injecting 0.1 mL of SPF isolates/eggs through the allantoic cavity. Each isolate was placed in five SPF eggs and then incubated in an incubator at 37 C and candled every day. Since day 2-4 post inoculation, embryo death has occurred. The eggs are harvested by their allantoic fluid and tested for haemagglutination test(HA/HI). The HI test results were confirmed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using the front primer FPHA232_13 (ATTGGTTAYCATGCAAAYAACTCG) and the back primer BPHA232_597 (GGAAAYATAGGTRGTTGGRTTYTGATAG) The results were five of the seven isolates were positive AI subtype B5 585 - 581 The five isolates of AI subtype H5N1 were subsequently sequenced, the results were all positive for AI virus subtype H5N1 clade 2.3.2. Each field isolate was given the name A / Chicken / Bali3A / GAY / 2019; A / Chicken / Bali9C / GAY / 2019; A / Chicken / BaliA4 / GAY / 2019; A / Chicken / Bali10A / GAY / 2016 and A / Chicken / Bali10C / GAY / 2019. One A / Chicken / Bali 9C / GAY / 2016 isolate was subsequently repeated 7 times until a stable H5N1 subtype AI virus titer was obtained. The results of matching with bioinformatics turned out that A / Chicken / Bali 9C / GAY / 2016 isolates had a kinship of 98.62% with AI subtype H5N1 Banyuwangi, amounting to 98.45% with AI subtype H5N1 Lamongan, amounting to 98.10% with AI-H5N1 Lumajang, 97.58% with AI-H5N1 Kediri, 97.07% with AIH5N1 Blitar, 96.72% with AI-H5N1 Denpasar, 96.72% with AI-H5N1 Buleleng and 96.72% with AI-H5N1 Sukoharjo. The conclusion is one of isolate namely A / Chicken / Bali 9C / GAY / 2019 including AI subtype H5N1 clade 2.3.2, is’t stable at passage on SPF eggs, has a kinship of 96.72% with A / duck / Sukoharjo / BBVW-1428- 9/2012, the virus content is 106.9 ELD50 so it is potential for vaccine candidates.

Evolution of H9N2 avian influenza viruses in Iran, 2017-2019.

Since its first detection in 1998, avian influenza virus (AIV) subtype H9N2 has been enzootic in Iran. To better understand the evolutionary history of H9N2 viruses in Iran, we sequenced 15 currently circulating H9N2 viruses from domestic poultry during 2017-2019 and performed phylogenetic analysis of complete genome sequences. Phylogenetic analyses indicated that the Iranian H9N2 viruses formed multiple well-supported monophyletic groups within the G1-lineage of H9N2 virus. Our analysis of viral population dynamics revealed an increase in genetic diversity until 2007, corresponding to the multiple introductions and diversification of H9N2 viruses into multiple genetic groups (named Iran 1-4 subgroups), followed by a sudden decrease after 2008. Only the Iran 4 subgroup has survived, expanded, and currently circulates in Iran. The H9N2 viruses possessed many molecular markers associated with mammalian adaption in all gene segments, except neuraminidase gene. Considering the presence of mammalian host-specific markers, the public health threat of H9N2 viruses continues. Molecular analysis showed that Iranian H9N2 strains have continued to evolve and recent strains have multiple amino acid changes and addition of potential N-glycosylation on the antigenic sites of haemagglutinin. Continued antigenic and molecular surveillance of H9N2 viruses in poultry and mammals would be required to monitor further increments in viral evolution and their potential threat to public health.

Establishing a Multicolor Flow Cytometry to Characterize Cellular Immune Response in Chickens Following H7N9 Avian Influenza Virus Infection.

Avian influenza virus (AIV) emerged and has continued to re-emerge, continuously posing great threats to animal and human health. The detection of hemagglutination inhibition (HI) or virus neutralization antibodies (NA) is essential for assessing immune protection against AIV. However, the HI/NA-independent immune protection is constantly observed in vaccines’ development against H7N9 subtype AIV and other subtypes in chickens and mammals, necessitating the analysis of the cellular immune response. Here, we established a multi-parameter flow cytometry to examine the innate and adaptive cellular immune responses in chickens after intranasal infection with low pathogenicity H7N9 AIV. This assay allowed us to comprehensively define chicken macrophages, dendritic cells, and their MHC-II expression, NK cells, γδ T cells, B cells, and distinct T cell subsets in steady state and during infection. We found that NK cells and KUL01+ cells significantly increased after H7N9 infection, especially in the lung, and the KUL01+ cells upregulated MHC-II and CD11c expression. Additionally, the percentages and numbers of γδ T cells and CD8 T cells significantly increased and exhibited an activated phenotype with significant upregulation of CD25 expression in the lung but not in the spleen and blood. Furthermore, B cells showed increased in the lung but decreased in the blood and spleen in terms of the percentages or/and numbers, suggesting these cells may be recruited from the periphery after H7N9 infection. Our study firstly disclosed that H7N9 infection induced local and systemic cellular immune responses in chickens, the natural host of AIV, and that the flow cytometric assay developed in this study is useful for analyzing the cellular immune responses to AIVs and other avian infectious diseases and defining the correlates of immune protection.