Pathogenic Avian Influenza H5N1 Virus Research Articles

Distinct but connected avian influenza virus activities in wetlands and live poultry markets in Bangladesh, 2018-2019.

From April 2018 to October 2019, we continued active surveillance for influenza viruses in Bangladeshi live poultry markets (LPMs) and in Tanguar Haor, a wetland region of Bangladesh where domestic ducks have frequent contact with migratory birds. The predominant virus subtypes circulating in the LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses of the H5N1-R1 genotype, like those found in previous years. Viruses of the H5N1-R2 genotype, which were previously reported as co-circulating with H5N1-R1 genotype viruses in LPM, were not detected. In addition to H9N2 viruses, which were primarily found in chicken and quail, H2N2, H3N8 and H11N3 LPAI viruses were detected in LPMs, exclusively in ducks. Viruses in domestic ducks and/or wild birds in Tanguar Haor were more diverse, with H1N1, H4N6, H7N1, H7N3, H7N4, H7N6, H8N4, H10N3, H10N4 and H11N3 detected. Phylogenetic analyses of these LPAI viruses suggested that some were new to Bangladesh (H2N2, H7N6, H8N4, H10N3 and H10N4), likely introduced by migratory birds of the Central Asian flyway. Our results show a complex dynamic of viral evolution and diversity in Bangladesh based on factors such as host populations and geography. The LPM environment was characterised by maintenance of viruses with demonstrated zoonotic potential and H5N1 genotype turnover. The wetland environment was characterised by greater viral gene pool diversity but a lower overall influenza virus detection rate. The genetic similarity of H11N3 viruses in both environments demonstrates that LPM and wetlands are connected despite their having distinct influenza ecologies.

The highly pathogenic H5N1 avian influenza virus induces the mitogen-activated protein kinase signaling pathway in the trachea of two Ri chicken lines.

ObjectiveThe highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the mitogen-activated protein kinase (MAPK) signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines.MethodsResistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, the tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing.ResultsA gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. A total of 1,794 DEGs were observed between control and H5N1-infected resistant Ri chickens, 432 DEGs between control and infected susceptible Ri chickens, and 1,202 DEGs between infected susceptible and infected resistant Ri chickens. The expression levels of MAPK signaling pathway-related genes (including MyD88, NF-κB, AP-1, c-fos, Jun, JunD, MAX, c-Myc), cytokines (IL-1β, IL-6, IL-8), type I interferons (IFN-α, IFN-β), and IFN-stimulated genes (Mx1, CCL19, OASL, and PRK) were higher in H5N1-infected than in non-infected resistant Ri chickens. MyD88, Jun, JunD, MAX, cytokines, chemokines, IFNs, and IFN-stimulated expressed genes were higher in resistant-infected than in susceptible-infected Ri chickens.ConclusionResistant Ri chickens showed higher antiviral activity compared to susceptible Ri chickens, and H5N1-infected resistant Ri chickens had immune responses and antiviral activity (cytokines, chemokines, interferons, and IFN-stimulated genes), which may have been induced through the MAPK signaling pathway in response to H5N1 infection.

Epizootic, Endemic and Pandemic Zoonotic Viral Infections

More than 60% human infectious diseases have zoonotic origin. Cross species transmission of pathogens is a continuous, dynamic process that occurs throughout the world, giving rise to epizootic (temporary, limited infection), endemic (on-going infection limited to a defined geographic region) and pandemic viral infections (infection spreading to every part of the world) like the current COVID-19 pandemic, which depends on the existing conditions on the ground. In Nov 2021, sudden mortality of numerous migrating demoiselle cranes was reported from their resting site near Jodhpur, Rajasthan. The symptomatic cranes became gradually weak and were unable to fly. They eventually fell dead which caused concern locally, given the current prevailing SARS-CoV-2 pandemic situation the world over. By the end of Dec 2021, the number of cranes with mortality and morbidity reduced, making it a temporary “epizootic infection”. Molecular diagnosis carried out at a specialized laboratory identified the etiological agent to be the highly pathogenic Avian Influenza Virus H5N1 (HPAIV), which has been responsible for morbidity of avian species from different parts of the world. There was no report of spreading the H5N1 AIV infection from the infected migratory cranes to nearby chicken farms or pig farms for now. In the absence of vaccines against the highly pathogenic H5N1 AIVs, and the inherent ability of influenza viruses, both avian AIV and human IAVs to constantly mutate its envelope gene or the surface antigens, resulting from the error-prone nature of the viral RNA Polymerase enzyme are the roadblocks for development of a universal, broad-spectrum influenza vaccine. Even when such a universal vaccine against H5N1 is available, vaccinating a large number of wild migratory cranes would be difficult. However, it is possible and indeed necessary to vaccinate chickens in poultry farms and pigs in farms that raise pigs for human consumption.

Immunogenicity and Cross-Protective Efficacy of Recombinant H5ha1 Protein of Clade 2.3.2.1a Highly Pathogenic H5n1 Avian Influenza Virus Expressed in E.Coli

Immunogenicity and Cross-Protective Efficacy of Recombinant H5ha1 Protein of Clade 2.3.2.1a Highly Pathogenic H5n1 Avian Influenza Virus Expressed in E.Coli

Improving siRNA design targeting nucleoprotein gene as antiviral against the Indonesian H5N1 virus

BackgroundSmall interfering RNA technology has been considered a prospective alternative antiviral treatment using gene silencing against influenza viruses with high mutations rates. On the other hand, there are no reports on its effectiveness against the highly pathogenic avian influenza H5N1 virus isolated from Indonesia.ObjectivesThe main objective of this study was to improve the siRNA design based on the nucleoprotein gene (siRNA-NP) for the Indonesian H5N1 virus.MethodsThe effectiveness of these siRNA-NPs (NP672, NP1433, and NP1469) was analyzed in vitro in Marbin-Darby canine kidney cells.ResultsThe siRNA-NP672 caused the largest decrease in viral production and gene expression at 24, 48, and 72 h post-infection compared to the other siRNA-NPs. Moreover, three serial passages of the H5N1 virus in the presence of siRNA-NP672 did not induce any mutations within the nucleoprotein gene.ConclusionsThese findings suggest that siRNA-NP672 can provide better protection against the Indonesian strain of the H5N1 virus.

Molecular Characterization of Avian Influenza H9N2 and H5N1 from Outbreak in a Backyard Layer Farm in Kaduna State, Nigeria

Avian influenza viruses (AIVs) have been reported in Nigeria since 2006, and the co-circulation of high-pathogenic avian influenza H5N1 and low-pathogenic avian influenza H9N2 subtypes in poultry has been reported. These Avian Influenza virus subtypes could lead to severe economic losses to the poultry farmers and may force the farmers out of the poultry production leading to unemployment for the staff of the farm. The co-infection with both highly pathogenic (H5N1) and low pathogenic (H9N2) could lead to possible re-assortment and the production of novel strain that can be highly pathogenic to chicken and may have the capacity to infect humans and be transmitted from human to humans which may lead to a pandemic of influenza. The clinical signs observed were somnolence, marginal cyanosis of the combs, ruffled feathers, matted vent and whitish/yellowish diarrhoea; while the post mortem examinations revealed dehydrated carcass, multiple foci of petechial haemorrhages on the abdominal fats, congested and necrotic-friable liver, edematous and congested lungs, congested spleen with multiple foci of necrosis, hemorrhagic and misshaped ovarian follicles, egg yolk peritonitis, swollen and hemorrhagic kidneys, erosion of the proventricular mucosa, congested trachea with mucoid exudates, and sinusitis. The disease was diagnosed based on the clinical signs, post mortem findings, competitive Enzyme-Linked Immunosorbent Assays (ELISAs) and clade-specific RT-qPCR, for the detection of co-circulating highly pathogenic avian influenza virus H5N1 and low pathogenic H9N2. The result of all sera (5/5) tested for Antibodies against Avian influenza Nucleoprotein produced positive results while 80 % of sera (4/5) tested positive for the H9 subtype of AIV. Antibodies against the H5 and H7 subtypes were not detected. The results of RRT-PCR and RRTPCR/ H5 were positive for both tracheal and cloacal samples. RRT-PCR/ H7 & H9 yielded negative results.

SUMOylation of Matrix Protein M1 and Filamentous Morphology Collectively Contribute to the Replication and Virulence of Highly Pathogenic H5N1 Avian Influenza Viruses in Mammals.

ABSTRACTThe matrix protein (M1) of influenza A virus plays an important role in replication, assembly, and budding. A previous study found that aspartic acid (D) at position 30 and alanine (A) at position 215 of M1 contribute to the high pathogenicity of H5N1 viruses in mice, and double mutations of D to asparagine (N) at position 30 (D30N) and A to threonine (T) at position 215 (A215T) in M1 dramatically attenuate H5N1 viruses in mice. However, the underlying mechanisms by which these M1 mutations attenuate the virulence of H5N1 viruses are unknown. Here, we found that the amino acid mutation A215T eliminates the SUMOylation of M1 by reducing its interaction with the host SUMO1 protein, significantly reducing the stability of M1, slowing the export of the M1-vRNP complex from the nucleus to the cytoplasm, and reducing viral replication in MDCK cells. We further found that the D30N mutation in M1 alters the shape of progeny viruses from filamentous to spherical virions. Our findings reveal an essential role for M1 215A SUMOylation and M1 30D-related filamentous morphology in the pathogenesis of avian influenza viruses, which could be targeted in novel antiviral drug designs.IMPORTANCE Identification of the pathogenic mechanism of highly pathogenic avian influenza viruses in mammals is helpful to develop novel anti-influenza virus strategies. Two amino acid mutations (D30N and A215T) in M1 were found to collectively attenuate H5N1 influenza viruses in mice, but the underlying mechanism remained unknown. This study found that the A215T mutation significantly decreases the SUMOylation of M1, which in turn attenuates the replication of H5N1 virus in mammalian cells. The D30N mutation in M1 was found to change the virion shape from filamentous to spherical. These findings are important for understanding the molecular mechanism of virulence of highly pathogenic avian influenza viruses in mammals.

Safety, Immunogenicity, and Protective Efficacy of an H5N1 Chimeric Cold-Adapted Attenuated Virus Vaccine in a Mouse Model

H5N1 influenza virus is a threat to public health worldwide. The virus can cause severe morbidity and mortality in humans. We constructed an H5N1 influenza candidate virus vaccine from the A/chicken/Guizhou/1153/2016 strain that was recommended by the World Health Organization. In this study, we designed an H5N1 chimeric influenza A/B vaccine based on a cold-adapted (ca) influenza B virus B/Vienna/1/99 backbone. We modified the ectodomain of H5N1 hemagglutinin (HA) protein, while retaining the packaging signals of influenza B virus, and then rescued a chimeric cold-adapted H5N1 candidate influenza vaccine through a reverse genetic system. The chimeric H5N1 vaccine replicated well in eggs and the Madin-Darby Canine Kidney cells. It maintained a temperature-sensitive and cold-adapted phenotype. The H5N1 vaccine was attenuated in mice. Hemagglutination inhibition (HAI) antibodies, micro-neutralizing (MN) antibodies, and IgG antibodies were induced in immunized mice, and the mucosal IgA antibody responses were detected in their lung lavage fluids. The IFN-γ-secretion and IL-4-secretion by the mouse splenocytes were induced after stimulation with the specific H5N1 HA protein. The chimeric H5N1 candidate vaccine protected mice against lethal challenge with a wild-type highly pathogenic avian H5N1 influenza virus. The chimeric H5 candidate vaccine is thus a potentially safe, attenuated, and reassortment-incompetent vaccine with circulating A viruses.

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

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

Amino acid substitutions in the H5N1 avian influenza haemagglutinin alter pH of fusion and receptor binding to promote a highly pathogenic phenotype in chickens.

Highly pathogenic H5N1 avian influenza viruses cause devastating outbreaks in farmed poultry with serious consequences for animal welfare and economic losses. Zoonotic infection of humans through close contact with H5N1 infected birds is often severe and fatal. England experienced an outbreak of H5N1 in turkeys in 1991 that led to thousands of farmed bird mortalities. Isolation of clonal populations of one such virus from this outbreak uncovered amino acid differences in the virus haemagglutinin (HA) gene whereby the different genotypes could be associated with distinct pathogenic outcomes in chickens; both low pathogenic (LP) and high pathogenic (HP) phenotypes could be observed despite all containing a multi-basic cleavage site (MBCS) in the HA gene. Using reverse genetics, three amino acid substitutions in HA were examined for their ability to affect pathogenesis in the chicken. Restoration of amino acid polymorphisms close to the receptor binding site that are commonly found in H5 viruses only partially improved viral fitness in vitro and in vivo. A third novel substitution in the fusion peptide, HA2G4R, enabled the HP phenotype. HA2G4R decreased the pH stability of HA and increased the pH of HA fusion. The substitutions close to the receptor binding site optimised receptor binding while modulating the pH of HA fusion. Importantly, this study revealed pathogenic determinants beyond the MBCS.

Substitution of I222L-E119V in neuraminidase from highly pathogenic avian influenza H7N9 virus exhibited synergistic resistance effect to oseltamivir in mice

That the high frequency and good replication capacity of strains with reduced susceptibility to neuraminidase inhibitors (NAIs) in highly pathogenic avian influenza H7N9 (HPAI H7N9) virus made it a significance to further study its drug resistance. HPAI H7N9 viruses bearing NA I222L or E119V substitution and two mutations of I222L-E119V as well as their NAIs-sensitive counterpart were generated by reverse genetics for NA inhibition test and replication capability evaluation in vitro. The attenuated H7N9/PR8 recombinant viruses were developed to study the pathogenicity and drug resistance brought by the above substitutions to mice. The IC50 fold change of oseltamivir to HPAI H7N9 with NA222L-119V is 306.34 times than that of its susceptible strain, and 3.5 times than the E119V mutant virus. HPAI H7N9 bearing NA222L-119V had good replication ability with peak value of more than 6log10 TCID50/ml in MDCK cells. H7N9/PR8 virus bearing NA222L-119V substitutions leaded to diffuse pneumonia, significant weight loss and fatality in mice. NA E119V made H7N9/PR8 virus resistant to oseltamivir, and I222L-E119V had synergistic resistance to oseltamivir in mice. Due to the good fitness of drug resistant strains of HPAI H7N9 virus, it is necessary to strengthen drug resistance surveillance and new drug research.

Dynamics of influenza A (avian influenza) virus in poultry in the Greater Accra region of Ghana amongst the production levels

ABSTRACT This research explores, through active surveillance, influenza A prevalence at different production levels in the Greater Accra region of Ghana, a study area with previous outbreak of highly pathogenic avian influenza H5N1 virus. The prevalence of influenza A was determined by rtRTPCR. This was achieved by screening 2040 samples comprising tracheal and cloacal swabs from chicken, ducks, pigeons, guinea fowls, and turkeys. Influenza A prevalence by production levels and species was computed at 95% confidence interval (CI) using the exact binomial interval. Structured questionnaires were also administered to 50 randomly selected poultry traders in the live bird markets. The overall influenza A prevalence was 7.7% (95% CI, 6.6, 8.9). Live bird market recorded 13.5% (n = 139, 95% CI, 11.5, 15.7), backyard poultry was 1.4% (95% CI, 0.6, 2.7), and commercial poultry 2.4% (95% CI, 1.2, 4.3). There was evidence of influenza A in all the poultry species sampled except for turkey. Subtyping of the M-gene has revealed the circulation of H9 in the three production levels. Live bird market has demonstrated high prevalence coupled with low level of biosecurity consciousness among the poultry operators. This is suggestive of live bird market serving as a potential basket for genetic reassortment with unpredictable future consequences.

Discrepancies in the efficacy of H5 inactivated avian influenza vaccines in specific-pathogen-free chickens against challenge with the Egyptian H5N8 clade 2.3.4.4 Group B virus isolated in 2018.

Background and Aim:Highly pathogenic avian influenza H5N8 virus of clade 2.3.4.4 was newly emerged to Egypt and firstly detected in carcasses of wild birds in November 2016. This study assessed the protection efficacy and virus shedding reduction of three different inactivated avian influenza (AI) H5 (H5N1, H5N2, and H5N3) commercial vaccines against challenge with two newly emerging highly pathogenic AI virus H5N8 Egyptian isolates in specific-pathogen-free (SPF) chicks.Materials and Methods:10-day-old SPF chicks (n=260) were divided into 20 groups (n=13). Groups 1-5 were vaccinated through the subcutaneous route (S/C) with 0.5 mL of H5N1 vaccine, Groups 6-10 were vaccinated (S/C) with 0.5 mL of H5N2 vaccine, and Groups 11-15 were vaccinated (S/C) with 0.5 mL of H5N3 vaccine. Positive control groups (16-19) were challenged at 25 and 31 days old (2 and 3 weeks post-vaccination [PV]) using H5N8 clade 2.3.4.4 A/duck/Egypt/F13666A/2017(H5N8) and H5N8 clade 2.3.4.4 A/chicken/Egypt/18FL6/2018(H5N8). Group 20 was left non-vaccinated as a control. All vaccinated groups were divided and challenged with both viruses at 25 and 31 days of age. The viral challenge dose was 0.1 mL of 106 EID50/0.1 mL titer/chick, and it was administered oronasally. All chicks were kept in isolators for 14 days after each challenge. Sera samples were collected weekly and at 2 weeks post-challenge (PC) to detect a humoral immune response. PC mortalities were recorded daily for 10 days to calculate the protection percentages. Tracheal swabs were collected from the challenged chicks in different groups at 3, 5, 7, and 10 days PC. Kidneys and spleens were collected at 3, 5, 7, and 10 days PC and kept in formalin for histopathological examination to assess lesions and severity scores. Tracheal swabs were inoculated in 10-day-old SPF embryonated chicken eggs for virus titration and to calculate shedding levels.Results:All studied vaccines displayed 70-100% protection within 10 days PC. Hemagglutination inhibition results from sera samples revealed antibody titers ranging from 0.6 to 5.4 log2 starting at 1-week PV with the highest titers at 4 weeks PV. Challenged SPF chickens exhibited a notable reduction in virus shedding, with an average of 1.5-2 log10, compared to control birds. Various histopathological lesions with different scores were detected.Conclusion:Our findings suggest that the inadequate virus shedding reduction and protection efficacy of studied vaccines were variable and that the type of vaccine to be used under field conditions should be reconsidered. Study of the variability between the Egyptian old emerged AI (AIV) 2017 H5N8 strains and the new emerging AIV 2018 H5N8 is required to achieve optimal protection and limit the current economic losses.

Inferring within-flock transmission dynamics of highly pathogenic avian influenza H5N8 virus in France, 2020.

Following the emergence of highly pathogenic avian influenza (H5N8) in France in early December 2020, we used duck mortality data from the index farm to investigate within‐flock transmission dynamics. A stochastic epidemic model was fitted to the daily mortality data and model parameters were estimated using an approximate Bayesian computation sequential Monte Carlo (ABC‐SMC) algorithm. The model predicted that the first bird in the flock was infected 5 days (95% credible interval, CI: 3–6) prior to the day of suspicion and that the transmission rate was 4.1 new infections per day (95% CI: 2.8–5.8). On average, ducks became infectious 4.1 h (95% CI: 0.7–9.1) after infection and remained infectious for 4.3 days (95% CI: 2.8–5.7). The model also predicted that 34% (50% prediction interval: 8%–76%) of birds would already be infectious by the day of suspicion, emphasizing the substantial latent threat this virus could pose to other poultry farms and to neighbouring wild birds. This study illustrates how mechanistic models can help provide rapid relevant insights that contribute to the management of infectious disease outbreaks of farmed animals. These methods can be applied to future outbreaks and the resulting parameter estimates made available to veterinary services within a few hours.

Site-Specific Glycan-Masking/Unmasking Hemagglutinin Antigen Design to Elicit Broadly Neutralizing and Stem-Binding Antibodies Against Highly Pathogenic Avian Influenza H5N1 Virus Infections.

The highly pathogenic avian influenza (HPAI) H5N1 viruses with the capability of transmission from birds to humans have a serious impact on public health. To date, HPAI H5N1 viruses have evolved into ten antigenically distinct clades that could cause a mismatch of vaccine strains and reduce vaccine efficacy. In this study, the glycan masking and unmasking strategies on hemagglutinin antigen were used for designing two antigens: H5-dm/st2 and H5-tm/st2, and investigated for their elicited immunity using two-dose recombinant H5 (rH5) immunization and a first-dose adenovirus vector prime, followed by a second-dose rH5 protein booster immunization. The H5-dm/st2 antigen was found to elicit broadly neutralizing antibodies against different H5N1 clade/subclade viruses, as well as more stem-binding antibodies to inhibit HA-facilitated membrane fusion activity. Mice immunized with the H5-dm/st2 antigen had a higher survival rate when challenged with homologous and heterologous clades of H5N1 viruses. Mutant influenza virus replaced with the H5-dm/st2 gene generated by reverse genetics (RG) technology amplified well in MDCK cells and embryonated chicken eggs. Again, the inactivated H5N1-dm/st2 RG virus elicited more potent cross-clade neutralizing and anti-fusion antibodies in sera. Therefore, the H5N1-dm/st2 RG virus with the site-specific glycan-masking on the globular head and the glycan-unmasking on the stem region of H5 antigen can be used for further development of cross-protective H5N1 vaccines.

Qualitative Risk Analysis of the Transmission of Highly Pathogenic Avian Influenza (HPAI) H5N1 through Manure Trade in Côte d'Ivoire

The contamination with Highly Pathogenic Avian Influenza (HPAI) H5N1 viruses occurs via the digestive tract following the ingestion of water or food contaminated with droppings of asymptomatic carriers or sick birds. Regarding the local practice of the use of poultry manure as an agricultural fertilizer, this study focuses on the risk of spread of the HPAI through the manure trade pathway in Côte d'Ivoire. For this purpose, epidemiological data and 96 poultry droppings samples were collected from 18 farms. The droppings samples were tested using the real time polymerase chain reaction (RT-PCR). The qualitative risk assessment (QRA) took into account event patterns by integrating all the pathways involved in the spread of HPAI. From the diagnostic test, all the 96 samples tested negative. Further investigations revealed that 74% of the Agnibilékrou farms experienced HPAI outbreaks in the past two years. The main risk factors identified were the movement of people, animals and fomites from one infected area to another. Additionally, the duration of storage of droppings and the distance between stockpiles and farms were potential risk factors. The QRA identified two levels of risk: moderate to high (60%) and low to negligible (40%). The estimated high risk occurs when the dropping is fresh and is low after an optimal period of storage. It is therefore necessary to make storage systematic and mandatory as a measure of treatment before the adoption of other complex measures such as composting and industrial processing.

EFFICACY OF GREEN TEA EXTRACT (GTE) AGAINST HIGHLY PATHOGENIC H5N1 AVIAN INFLUENZA VIRUS; IN VIVO AND IN VITRO STUDY

Highly pathogenic avian influenza virus (HPAIV) H5N1 is an endemic disease in Egypt and severe outbreaks have been reported even in vaccinated flocks. Several H5 vaccines have failed to protect birds against Egyptian H5N1 virus. In this study, the antiviral effect of green tea extract (GTE) in combination with inactivated H5N1 vaccine was evaluated. GTE found to be safe at high concentration on Vero cells and decrease the CPE on infected cells. In vivo, specific pathogen free chickens were allocated into four groups, 15 birds each. Negative control group, positive control group, vaccinated group, vaccinated group supplied by GTE in drinking water (5gm/L). Blood samples were collected weekly and tested using (HI) test. Lung tissue specimens collected for histopathology and immunohistochemistry. Interestingly, vaccinated non-treated birds exhibited severe post vaccinal reaction and deaths. GTE in combination with inactivated vaccine increased HI titer one-week post vaccination, and no deaths were recorded in this group. Moreover, treated chickens did not excrete virus in swab samples and minimal antigen and pathological changes were reported in its lungs. The addition of GTE prevent the shedding of the virus and achieve complete protection against HPAI.

Protective Efficacy Evaluation of Four Inactivated Commercial Vaccines Against Low Pathogenic Avian Influenza H9N2 Virus Under Experimental Conditions in Broiler Chickens.

Avian influenza vaccines are commonly used in the poultry industry. The objective of this study was to compare, under experimental conditions, the protective efficacy of four imported commercial inactivated H9N2 vaccines (A, B, C, and D) in broiler chickens. A total of 150 one-day-old chicks were divided into six groups: four experimental groups, each containing 30 chicks, received one of the vaccines (A, B, C, or D) delivered in a 0.3-ml dose subcutaneously at 1 day of age, whereas the control, Group T, was not vaccinated but challenged and Group E was kept unvaccinated and unchallenged. At 21 days postvaccination, Groups A, B, C, D, and T were challenged with 107 embryo infective dose 50% of A/Chicken/Morocco/01/2016 (H9N2). All chicks were observed daily for clinical signs during the 12 days postchallenge (dpc). At 5 and 12 dpc, chicks were euthanatized for necropsy examination. Blood samples were collected weekly for serologic analysis and oropharyngeal swabs were collected for virus detection by real-time RT-PCR. Respiratory signs started at 48 hr pc and maximum severity was observed on 9 dpc. Chiefly, the birds vaccinated with vaccine B showed significantly more respiratory signs than did their counterparts. Serologic analysis revealed that the sera of Groups A and D birds showed a decrease in antibody (Ab) levels up to day 26; then a slight increase of Ab level was observed until day 31, while Group B and C birds showed a stabilization of the titers from day 21 until the end of the experiment. The viral shedding rate was significantly lower in Groups C and A (40%-50% of the birds shed virus for <7 days) compared with other challenged groups (60%-75% of the birds shed virus for ≥9 days). This experiment illustrated that vaccination applied on the first day in the hatchery with the four vaccines tested did not provide an acceptable protection against H9N2 in comparison with the controls that did not receive any vaccine. However, at first glance, we might favor vaccines A and C for their ability to reduce and shorten viral shedding as compared with vaccines B and D.

Optimizing the early detection of low pathogenic avian influenza H7N9 virus in live bird markets.

In Southeast Asia, surveillance at live bird markets (LBMs) has been identified as crucial for detecting avian influenza viruses (AIV) and reducing the risk of human infections. However, the design of effective surveillance systems in LBMs remains complex given the rapid turn-over of poultry. We developed a deterministic transmission model to provide guidance for optimizing AIV surveillance efforts. The model was calibrated to fit one of the largest LBMs in northern Vietnam at high risk of low pathogenic H7N9 virus introduction from China to identify the surveillance strategy that optimizes H7N9 detection. Results show that (i) using a portable diagnostic device would slightly reduce the number of infected birds leaving the LBM before the first detection, as compared to a laboratory-based diagnostic strategy, (ii) H7N9 detection could become more timely by sampling birds staying overnight, just before new susceptible birds are introduced at the beginning of a working day, and (iii) banning birds staying overnight would represent an effective intervention to reduce the risk of H7N9 spread but would decrease the likelihood of virus detection if introduced. These strategies should receive high priority in Vietnam and other Asian countries at risk of H7N9 introduction.

Impact of Individual Viral Gene Segments from Influenza A/H5N8 Virus on the Protective Efficacy of Inactivated Subtype-Specific Influenza Vaccine

Since its emergence in 2014, the highly pathogenic avian influenza H5N8 virus has continuously and rapidly spread worldwide in the poultry sector resulting in huge economic losses. A typical inactivated H5N8 vaccine is prepared using the six internal genes from A/PR8/1934 (H1N1) and the two major antigenic proteins (HA and NA) from the circulating H5N8 strain with the HA modified to a low pathogenic form (PR8HA/NA-H5N8). The contribution of the other internal proteins from H5N8, either individually or in combination, to the overall protective efficacy of PR8-based H5N8 vaccine has not been investigated. Using reverse genetics, a set of PR8-based vaccines expressing the individual proteins from an H5N8 strain were rescued and compared to the parent PR8 and low pathogenic H5N8 strains and the commonly used PR8HA/NA-H5N8. Except for the PR8-based vaccine strains expressing the HA of H5N8, none of the rescued combinations could efficiently elicit virus-neutralizing antibodies. Compared to PR8, the non-HA viral proteins provided some protection to infected chickens six days post infection. We assume that this late protection was related to cell-based immunity rather than antibody-mediated immunity. This may explain the slight advantage of using full low pathogenic H5N8 instead of PR8HA/NA-H5N8 to improve protection by both the innate and the humoral arms of the immune system.