HPAI H5N1 Research Articles

Field efficacy of HVT-H9 vaccination after natural infection of H9N2 avian influenza virus in broilers

H9N2 subtype avian influenza virus (AIV) has been persistently circulating in China. It causes huge economic losses to the poultry industry and poses a great threat to public health. Previously, we constructed a turkey herpesvirus live vector vaccine candidate strain expressing an H9 gene, HVT-H9. Results showed that immunisation with HVT-H9 could provide good immunity in specific pathogen free (SPF) chickens. In this study, field-bred Arbour Acres plus (AA+) broilers were additionally immunised with HVT-H9 at one day of age. Then, broilers were naturally infected with H9N2 AIV. During the endemic period, death occurred in flocks without HVT-H9 immunisation and the mortality rate increased rapidly, forming a clear death wave. However, HVT-H9 vaccination prevented broiler mortality. Etiological tests and serological tests showed that broilers were positive for H9N2 AIV. Collectively, HVT-H9 immunisation provided good immunity for broilers in the field by inhibiting H9N2 virus infection and transmission.

Avian influenza (H5N1) virus, epidemiology and its effects on backyard poultry in Indonesia: a review.

Avian influenza (AI) is a zoonotic viral endemic disease that affects poultry, swine, and mammals, including humans. Highly pathogenic avian influenza (HPAI) is caused by influenza type A virus subtypes H5, and H7 which are naturally carried by a wild bird and often affect domestic poultry. Avian influenza (AI) is a major problem worldwide that causes significant economic losses in the poultry sector. Since 2003, the widespread H5N1 HPAI in poultry has led to high mortalities resulting in huge economic losses in the poultry sector in Indonesia. Domestic poultry is a key source of income that contributes to economic growth, both directly and indirectly, by reducing poverty among the people living in rural communities. Furthermore, in many developing countries, including Indonesia, rural people meet a portion of their food needs through backyard poultry. Nevertheless, this sector is strongly affected by biosecurity hazards, particularly in Indonesia by HPAI infections. Avian influenza (AI), subtype H5N1 has zoonotic significance, posing major risks to public health and poultry. Due to close interaction between wild migratory birds and ducks, the domestic poultry sector in Indonesia is directly affected by this virus. This virus continues to be ubiquitous in Indonesia as a result of the unpredictable mutations produced by antigenic drift and shift, which can persist from a few days to several years. In this review, the epidemiology and impact, of highly pathogenic avian influenza H5N1 subtype virus infection on backyard poultry in Indonesia were discussed.

PA-X protein of H9N2 subtype avian influenza virus suppresses the innate immunity of chicken bone marrow-derived dendritic cells

H9N2 subtype avian influenza (AI) is an infectious disease associated with immunosuppression in poultry. Here, the regulation function of PA-X protein was determined on the host innate immune response of H9N2-infected chicken bone marrow-derived DCs (chBM-DCs). Based on 2 mutated viruses expressing PA-X protein (rTX) or deficient PA-X protein (rTX-FS), and the established culture system of chBM-DCs, results showed PA-X protein inhibited viral replication in chBM-DCs but not in non-immune chicken cells (DF-1). Moreover, PA-X protein downregulated the expression of phenotypic markers (CD40, CD86, and MHCII) and proinflammatory cytokine (IL-12 and IL-1β) of chBM-DCs. The mixed lymphocyte reaction between chBM-DCs and chicken T cells showed PA-X protein significantly decreased H9N2-infected chBM-DCs to induce T cell proliferation, implying a suppression of the DC-induced downstream T cell response. Taken together, these findings indicated that PA-X protein is a key viral protein to help H9N2 subtype AIVs escape the innate immunity of chBM-DCs.

Phylodynamics of Highly Pathogenic Avian Influenza A(H5N1) Virus Circulating in Indonesian Poultry.

After its first detection in 1996, the highly pathogenic avian influenza A(H5Nx) virus has spread extensively worldwide. HPAIv A(H5N1) was first detected in Indonesia in 2003 and has been endemic in poultry in this country ever since. However, Indonesia has limited information related to the phylodynamics of HPAIv A(H5N1) in poultry. The present study aimed to increase the understanding of the evolution and temporal dynamics of HPAIv H5N1 in Indonesian poultry between 2003 and 2016. To this end, HPAIv A(H5N1) hemagglutinin sequences of viruses collected from 2003 to 2016 were analyzed using Bayesian evolutionary analysis sampling trees. Results indicated that the common ancestor of Indonesian poultry HPAIv H5N1 arose approximately five years after the common ancestor worldwide of HPAI A(H5Nx). In addition, this study indicated that only two introductions of HPAIv A(H5N1) occurred, after which these viruses continued to evolve due to extensive spread among poultry. Furthermore, this study revealed the divergence of H5N1 clade 2.3.2.1c from H5N1 clade 2.3.2.1b. Both clades 2.3.2.1c and 2.3.2.1b share a common ancestor, clade 1, suggesting that clade 2.3.2.1 originated and diverged from China and other Asian countries. Since there was limited sequence and surveillance data for the HPAIv A(H5N1) from wild birds in Indonesia, the exact role of wild birds in the spread of HPAIv in Indonesia is currently unknown. The evolutionary dynamics of the Indonesian HPAIv A(H5N1) highlight the importance of continuing and improved genomic surveillance and adequate control measures in the different regions of both the poultry and wild birds. Spatial genomic surveillance is useful to take adequate control measures. Therefore, it will help to prevent the future evolution of HPAI A(H5N1) and pandemic threats.

Mutation of D201G near the receptor binding site significantly drives antigenic drift of circulating H9N2 subtype avian influenza virus.

The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused significant losses in chicken flocks throughout China. Our previous research has shown that field isolates of H9N2 underwent antigenic drift to evolve into distinct groups with significant antigenic divergence from the commercially available vaccines. The present study sought to identify which single mutations that have naturally appeared in isolates from the past 5 years have driven antigenic drift. Six high-frequency mutation sites in/near the receptor binding site region were screened by comparing amino acid alignments of the H9N2 AIVs isolated from China between 2014 and 2019. Two substitutions (A168N and D201G) were demonstrated to have a significant impact on the antigenicity but did not change the growth kinetics of the virus. It is worth noting that the D201G substitution not only significantly changed the antigenicity but also caused immune escape against the parental virus. In conclusion, A168N and D201G substitution are newly discovered determinants that can significantly change the antigenicity of H9N2 AIV, which should be tracked during outbreaks.

Multiple independent introductions of highly pathogenic avian influenza H5 viruses during the 2020-2021 epizootic in France.

During winter 2020-2021, France and other European countries were severely affected by highly pathogenic avian influenza H5 viruses of the Gs/GD/96 lineage, clade 2.3.4.4b. In total, 519 cases occurred, mainly in domestic waterfowl farms in Southwestern France. Analysis of viral genomic sequences indicated that 3 subtypes of HPAI H5 viruses were detected (H5N1, H5N3, H5N8), but most French viruses belonged to the H5N8 subtype genotype A, as Europe. Phylogenetic analyses of HPAI H5N8 viruses revealed that the French sequences were distributed in 9 genogroups, suggesting 9 independent introductions of H5N8 from wild birds, in addition to the 2 introductions of H5N1 and H5N3.

Epidemiological investigation of infectious diseases in geese on mainland China during 2018-2021.

Geese play an important role in agricultural economics, with China producing the vast majority of goose meat consumed worldwide annually. The variations in the avian viruses and co-infections result in substantial economic losses to the goose industry in China. To understand the evolutionary characteristics and co-infections of viruses, a broad epidemiological investigation of epizootic viruses of goose was carried out in nine provinces of China during 2018-2021. Here, the results indicated that, among the 1970 clinical samples, 50.81% (1001/1970) were positive for goose astrovirus (GAstV), 18.22% (359/1970) for avian orthoreovirus, 12.74% (251/1970) for goose parvovirus, 11.02% (217/1970) for H9N2 subtype avian influenza virus, 4.01% (79/1970) for Newcastle disease virus, and 2.08% (41/1970) for fowl adenovirus. Among the six viruses, co-infections comprised a large proportion (66.37%) in the field, of which the dual infection was more common. Additionally, phylogenetic analysis of GAstVs indicated that Chinese GAstVs had formed two distinct groups, that is, GAstV-1 and GAstV-2. GAstV-2 sub-genotype II-c had arisen as the dominant genotype in the whole country. Notably, all the H9N2-AIV isolated strains harboured the mammalian adaptation markers I155T, H183N, and Q226L (H3 numbering) in the HA gene, which promotes preferential binding to human-like α2-6-linked sialic acid receptors. And beyond that, we determined that the goose-origin Muscovy Duck Reovirus isolates, showing 51.7%-96% similarities to that of other waterfowl-origin orthoreovirus isolates in sequence homology analysis of the representative part of σC, are a new variant of waterfowl-origin orthoreovirus. These data provide valuable information about the prevalence of infectious diseases in geese on mainland China.

The Function behind the Relation between Lipid Metabolism and Vimentin on H9N2 Subtype AIV Replication.

Avian influenza caused by H9N2 subtype avian influenza virus (AIV) poses a great threat to the healthy development of the poultry industry. Vimentin is closely related to intracellular lipid metabolism, which plays an important role during the viral infection process. However, the function of lipid metabolism and vimentin on H9N2 AIV replication is unclear. In this paper, the cholesterol level and 3-hydroxy-3-methylglutaryl coenzyme a reductase (HMGCR) phosphorylation were investigated in vimentin knockout (KO) and human cervical carcinoma cells (HeLa) cell with or without AIV infection. The results showed that compared to the control group without infected with H9N2 subtype AIV, the cholesterol contents were significantly increased, while HMGCR phosphorylation level was reduced in both KO and HeLa cell after virus infection. Furthermore, viral replication was significantly inhibited in the cells treated with the cholesterol inhibitor lovastatin. Compared with the control group, adenylate activated protein kinase (AMPK), a kinase regulating HMGCR enzymatic activity was inhibited in both KO and HeLa cells in the infected virus group, and AMPK phosphorylation levels were significantly lower in KO HeLa cell than that of HeLa cells. Additionally, after MβCD treatment, viral hemagglutinin (HA) gene level was significantly decreased in HeLa cells, while it was significantly increased in KO HeLa cells. In addition, vimentin expression was significantly increased in MβCD-treated HeLa cells with the viral infection and returned to normal levels after exogenous cholesterol to backfill the MβCD-treated cells. Therefore, the disruption of lipid rafts during the binding phase of viral invasion of cells significantly reduced viral infection. These studies indicated that the lipid rafts and cholesterol levels might be critical for H9N2 subtype AIV infection of human-derived cells and that vimentin might play an important role in the regulation of lipids on viral replication, which provided an important antiviral target against influenza virus.

PA-X protein assists H9N2 subtype avian influenza virus in escaping immune response of mucosal dendritic cells.

H9N2 subtype low pathogenicity avian influenza virus (AIV) poses a potential zoonotic risk. PA-X, a novel protein generated by PA gene ribosomal frameshift, is considered to be the virulence factor of H9N2 subtype AIVs. Our study found that rTX possessing PA-X protein enhanced the mammalian pathogenicity of H9N2 subtype AIVs compared with PA-X-deficient virus (rTX-FS). Furthermore, PA-X protein inhibited H9N2 subtype AIVs to infect dendritic cells (DCs), but not nonimmune cells (MDCK cells). Meanwhile, PA-X protein suppressed the phenotypic expression (CD80, CD86, CD40 and MHCII), early activation marker (CD69) and pro-inflammatory cytokines (IL-6 and TNF-α), whereas increased anti-inflammatory cytokine (IL-10) in DCs. After intranasally viral infection in mice, we found that PA-X protein of H9N2 subtype AIVs reduced CD11b+ and CD103+ subtype mucosal DCs recruitment to the nasal submucosa by inhibiting CCL20 expression. Moreover, PA-X protein abolished the migratory ability of CD11b+ and CD103+ DCs into draining cervical lymph nodes by down-regulating CCR7 expression. The rTX-infected DCs significantly impaired the allogeneic CD4+ T cell proliferation, suggesting PA-X protein suppressed the immune functions of DCs for hindering the downstream immune activation. These findings indicated that PA-X protein assisted H9N2 subtype AIVs in escaping immune response of mucosal DCs for enhancing the pathogenicity.

Efficacy of recombinant Newcastle disease virus expressing HA protein of H9N2 Avian influenza virus in respiratory and intestinal tract

H9N2 subtype avian influenza virus (AIV) is a low pathogenic AIV, which is widely prevalent all over the world. The infection of H9N2 AIV often leads to secondary infection with other pathogens, causing serious economic losses to poultry industry. Up to now, several recombinant Newcastle disease viruses (NDV) expressing H9N2 AIV hemagglutinin (HA) protein had been developed. However, the efficacy of recombinant virus on tracheal and intestinal injury caused by H9N2 AIV was rarely reported. The aim of this study was to evaluate the efficacy of recombinant NDV expressing H9N2 AIV HA protein in respiratory and intestinal tract. In this study, based on Red/ET homologous recombination technology, H9N2 AIV HA gene was embedded into the genome of NDV LaSota vaccine strain to obtain the recombinant virus rNDV-H9. The recombinant virus rNDV-H9 showed similar replication kinetic characteristics with the parent LaSota strain and had good genetic stability. The immunization result showed that rNDV-H9 induced high HI antibody titer against H9N2 AIV. In the H9N2 AIV challenge experiment, rNDV-H9 could significantly reduce the virus shedding in trachea and cloaca. In addition, rNDV-H9 protected the barrier function of chicken intestinal mucosal epithelial cells and reduced the virus-induced inflammatory response to a certain extent, so as to inhibit the abnormal proliferation of E. coli. This study suggests that rNDV-H9 is a promising vaccine candidate against H9N2 AIV.

Improved Whole Gamma Irradiated Avian Influenza Subtype H9N2 Virus Vaccine Using Trehalose and Optimization of Vaccination Regime on Broiler Chicken

Gamma (γ)-radiation can target viral genome replication and preserve viral structural proteins compared to formalin inactivation. Thus, a stronger immunity could be induced after the inoculation of the irradiated virus. In this study, γ-irradiated low-pathogenic avian influenza virus-H9N2 (LPAIV-H9N2) was used to immunize the broiler chicken in two formulations, including γ-irradiated LPAIV-H9N2 with 20% Trehalose intranasally (IVT.IN) or γ-irradiated LPAIV-H9N2 plus Montanide oil adjuvant ISA70 subcutaneously (IV+ISA.SC) in comparison with formalin-inactivated LPAIV-H9N2 vaccine intranasally (FV.IN) or formalin-inactivated LPAIV-H9N2 plus ISA70 subcutaneously (FV+ISA.SC). Two vaccination regimes were employed; the first one was primed on day 1 and boosted on day 15 (early regime), and the second one was primed on day 11 and boosted on day 25 (late regime). A challenge test was performed with a live homologous subtype virus. Virus shedding was monitored by quantifying the viral load via RT-qPCR on tracheal and cloacal swabs. Hemagglutination inhibition (HI) antibody titration and stimulation index (SI) of the splenic lymphocyte proliferation were measured, respectively, by HI test and Cell Proliferation assay. Cytokine assay was conducted by the RT-qPCR on antigen-stimulated spleen cells. The results of the HI test showed significant increases in antibody titer in all vaccinated groups, but it was more evident in the IVT late vaccination regime, reaching 5.33 log2. The proliferation of stimulated spleen lymphocytes was upregulated more in the IVT.IN vaccine compared to other vaccines. The mRNA transcription levels of T-helper type 1 cytokines such as interferon-gamma (IFN-γ) and interleukin 2 (IL-2) were upregulated in all vaccinated groups at the late regime. Moreover, IL-6, a pro-inflammatory cytokine was upregulated as well. However, upregulation was more noticeable in the early vaccination than the late vaccination (p< 0.05). After the challenge, the monitoring of virus shedding for the H9 gene represented an extremely low viral load. The body weight loss was not significant (p > 0.05) among the vaccinated groups. In addition, the viral load of <100.5 TCID50/ml in the vaccinated chicken indicated the protective response for all the vaccines. Accordingly, the IVT vaccine is a good candidate for the immunization of broiler chicken via the intranasal route at late regime.

Evaluation of immunostimulatory effects of a commercial herbal extract on avian influenza subtype H9N2 and Newcastle disease vaccination in chickens

Contrary to several vaccination programs to protect against bird flu and Newcastle disease (ND), these diseases remain a significant threat to the poultry industry. Improving the immune responses is important for many reasons such as vaccine failure, immunosuppressive diseases, and antibiotics misuse. Nowadays, research on materials likely to have immune-enhancing effects is increasing. Factors, substances, or compounds that can regulate different functions of the immune system can be used to enhance specific and even nonspecific responses to vaccines. The ability of herbal extracts to improve the immune system supports their use as immune stimulants. The present study aimed to examine the effects of barley malt extract in drinking water on humoral immunity of broiler chickens against ND and Avian Influenza (AI) disease subtype H9N2 vaccines. A total of 225 one-day-old broiler chicks (Ross strain) were divided into 5 groups of 3 subgroups and each subgroup had 15 chicks. Group A, B, and C chickens received 0.2%, 0.3%, and 0.5% of malt extract respectively in drinking water. Group D chickens did not get malt extract. Group E chickens did not receive malt extract and Newcastle and AI vaccines as the control group. All groups except group E were vaccinated with live Newcastle vaccine (B1 strain) intraocularly and AI-ND subtype H9N2 killed vaccine subcutaneously on the 7th day. Antibody titer against NDs and AI vaccines was considered by the Hemagglutination Inhibition test (HI test). Malt extract at 0.5% concentration, at all periods after vaccination, enhanced the systemic antibody response to ND vaccine in broiler chickens, but this extract had no significant effect on antibody response against the AI vaccine.Conclusion: Inoculation of ND vaccines with barley malt extract as an immune-boosting agent induces extensive immune responses involved in HI-NDV Ab titers.

Emerging threats and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review.

Avian influenza virus subtype H9N2 was first documented in Indonesia in 2017. It has become prevalent in chickens in many provinces of Indonesia as a result of reassortment in live bird markets. Low pathogenic avian influenza subtype H9N2 virus-infected poultry provides a new direction for the influenza virus. According to the latest research, the Indonesian H9N2 viruses may have developed through antigenic drift into a new genotype, posing a significant hazard to poultry and public health. The latest proof of interspecies transmission proposes that the next human pandemic variant will be the avian influenza virus subtype H9N2. Manipulation and elimination of H9N2 viruses in Indonesia, constant surveillance of viral mutation, and vaccine updates are required to achieve effectiveness. The current review examines should be investigates/assesses/report on the development and evolution of newly identified H9N2 viruses in Indonesia and their vaccination strategy.

Characterization of Hemagglutinin Gene Fragment of H9N2 Avian Influenza Virus Isolated from Environmental Live Bird Market in the Greater Jakarta Area

Virus H9N2 has become the most common subtype of low pathogenic avian influenza (LPAI) in poultry and shows the ability to infect humans. One of the important factors triggering the virus ​​pandemic is the live bird market (LBM). The virus acts as an internal gene donor in the other subtypes, such as H5N1, H5N2, H7N9, and H10N8, in poultry and humans. This study was conducted to detect the presence of the H9N2 virus and the molecular characteristics of the hemagglutinin gene fragment at the cleavage site, receptor binding site, antigenic site, and glycosylation site positions of the H9N2 subtype AI virus isolated from the LBM environment. This study used Disease Investigation Center Subang isolates from environmental samples of LBM in the Greater Jakarta area (DKI Jakarta, Bogor, Depok, Tangerang, and Bekasi) in 2019. Based on molecular detection using RT-PCR and RT-qPCR, it was found that avian influenza subtype H9N2 was detected. This indicates that LBM has the potential to be the source of the spread of the ​​H9N2 virus. The pattern of the amino acid cleavage site PSRSSR↓GLF indicates that the research sample belongs to the low pathogenic AI. The substitution of amino acids in the receptor binding site and antigenic site increases the specificity of non-human host recognition. The potential glycosylation site, with the NCS motif found in amino acid position 295-297, close to the receptor-binding site. Based on genetic analysis and phylogenetic topography, the virus is included in the CVI (China, Vietnam, Indonesia) lineage and subclade H9.4.2.5.

Emerging threat and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review.

Avian influenza virus subtype H9N2 was first documented in Indonesia in 2017. It has become prevalent in chickens in many provinces of Indonesia as a result of reassortment in live bird markets. Low pathogenic avian influenza subtype H9N2 virus-infected poultry provides a new direction for influenza virus. According to the latest research, the Indonesian H9N2 viruses may have developed through antigenic drift into new genotype, posing a significant hazard to poultry and public health. The latest proof of interspecies transmission proposes that, the next human pandemic variant will be avian influenza virus subtype H9N2. Manipulation and elimination of H9N2 viruses in Indonesia, constant surveillance of viral mutation, and vaccines updates are required to achieve effectiveness. The current review examines should be investigates/assesses/report on the development and evolution of newly identified H9N2 viruses in Indonesia and their vaccination strategy.

Hemagglutinin Gene Variation Rate of H9N2 Avian Influenza Virus by Vaccine Intervention in China.

H9N2 subtype avian influenza virus (AIV) is widespread globally, with China being the main epidemic center. Inactivated virus vaccination was adopted as the main prevention method in China. In this study, 22 hemagglutinin (HA) sequences were obtained from all inactivated vaccine strains of H9N2 subtype AIVs in China since its introduction. A phylogenetic analysis of the vaccine sequences and HA sequences of all published H9N2 subtype AIVs was conducted to investigate the relationship between vaccine use and the virus genetic diversity of the virus. We found that during 2002–2006, when fewer vaccines were used, annual genetic differences between the HA sequences were mainly distributed between 0.025 and 0.075 and were mainly caused by point mutations. From 2009 to 2013, more vaccines were used, and the genetic distance between sequences was about 10 times greater than between 2002 and 2006, especially in 2013. In addition to the accumulation of point mutations, insertion mutations may be the main reason for the large genetic differences between sequences from 2009 to 2013. These findings suggest that the use of inactivated vaccines affected point mutations in the HA sequences and that the contribution of high-frequency replacement vaccine strains to the rate of virus evolution is greater than that of low-frequency replacement vaccine strains. The selection pressure of the vaccine antibody plays a certain role in regulating the variation of HA sequences in H9N2 subtype AIV.

Genuine lethal infection of a wood pigeon (Columba palumbus) with high pathogenicity avian influenza H5N1, clade 2.3.4.4b, in Germany, 2022

Despite the increasing frequency of avian influenza (AI) cases in wild birds in Europe during the last decade, doves and pigeons were not recognized to be part of the AI epidemiology. Here we describe a natural, lethal HPAIV infection of subtype H5N1, clade 2.3.4.4b, in a wood pigeon (Columba palumbus) in Germany, 2022. The animal was housed in a bird sanctuary that suffered an HPAI-outbreak with multiple bird species affected. The pigeon’s post mortem findings were dominated by an acute lymphohistiocytic meningoencephalitis as well as neuronal necrosis in the grey matter of the cerebral hemispheres and in the brain stem. Influenza A viral antigen was associated to these alterations with a striking ependymal and periventricular distribution most probably indicating intraventicular liquorogenic spread of the virus. In addition, typical severe multifocal to coalescing necrotizing pancreatitis was evident. Occasionally, vascular endothelial cells showed an intense viral antigen staining. Examination of oropharyngeal and cloacal swabs and of various tissues by real-time RT-PCR corroborated systemic infection with highly pathogenic (HP) AIV of subtype H5N1. Viral loads soared in brain and pancreas samples. Full genome sequencing directly from brain tissue confirmed the assignment of the virus to clade 2.3.4.4b of the goose/Guangdong lineage bearing a polybasic hemagglutinin cleavage site. Our results demonstrate the principal susceptibility of wood pigeons to HPAIV H5N1 resulting in a sporadic infection. Considering the wide distribution and roaming of wild, feral, and racing pigeons with often close contact to humans, thorough investigation of suspected cases including pathological manifestation is advisable.

Spatiotemporal analysis of highly pathogenic avian influenza (H5N1) outbreaks in poultry in Egypt (2006 to 2017)

BackgroundIn Egypt, the highly pathogenic avian influenza (HPAI) subtype H5N1 is endemic and possesses a severe impact on the poultry. To provide a better understanding of the distributional characteristics of HPAI H5N1 outbreaks in Egypt, this study aimed to explore the spatiotemporal pattern and identify clusters of HPAI H5N1 outbreaks in Egypt from 2006 to 2017.ResultsThe Epidemic curve (EC) was constructed through time series analysis; in which six epidemic waves (EWs) were revealed. Outbreaks mainly started in winter peaked in March and ended in summer. However, newly emerged thermostable clades (2.2.1.1 and 2.2.1.2) during the 4th EW enabled the virus to survive and cause infection in warmer months with a clear alteration in the seasonality of the epidemic cycle in the 5th EW. The endemic situation became more complicated by the emergence of new serotypes. As a result, the EC ended up without any specific pattern since the 6th EW to now. The spatial analysis showed that the highest outbreak density was recorded in the Nile Delta considering it as the ‘Hot spot’ region. By the 6th EW, the outbreak extended to include the Nile valley. From spatiotemporal cluster epidemics, clustering in the Delta was a common feature in all EWs with primary clusters consistently detected in the hot-spot region, but the location and size varied with each EW. The highest Relative Risk (RR) regions in an EW were noticed to contain the primary clusters of the next EW and were found to include stopover sites for migratory wild birds. They were in Fayoum, Dakahlia, Qalyobiya, Sharkia, Kafr_Elsheikh, Giza, Behera, Menia, and BeniSuef governorates. Transmission of HPAI H5N1 occurred from one location to another directly resulted in a series of outbreaks forming neighboring secondary clusters. The absence of geographical borders between the governorates in addition to non-restricted movements of poultry and low vaccination and surveillance coverage contributed to the wider spread of infection all over Egypt and to look like one epidemiological unit.ConclusionOur findings can help in better understanding of the characteristics of HPAI H5N1 outbreaks and the distribution of outbreak risk, which can be used for effective disease control strategies.Graphical abstract

Maternal-derived antibodies hinder the antibody response to H9N2 AIV inactivated vaccine in the field

AbstractThe H9N2 subtype avian influenza virus (AIV) inactivated vaccine has been used extensively in poultry farms, but it often fails to stimulate a sufficiently high immune response in poultry in the field, although it works well in laboratory experiments; hence, the virus still causes economic damage every year and poses a potential threat to public health. Based on surveillance data collected in the field, we found that broilers with high levels of maternal-derived antibodies (MDAs) against H9N2 virus did not produce high levels of antibodies after vaccination with a commercial H9N2 inactivated vaccine. In contrast, specific pathogen-free (SPF) chickens without MDAs responded efficiently to that vaccination. When MDAs were mimicked by administering passively transferred antibodies (PTAs) into SPF chickens in the laboratory, similar results were observed: H9N2-specific PTAs inhibited humoral immunity against the H9N2 inactivated vaccine, suggesting that H9N2-specific MDAs might hinder the generation of antibodies when H9N2 inactivated vaccine was used. After challenge with homologous H9N2 virus, the virus was detected in oropharyngeal swabs of the vaccinated and unvaccinated chickens with PTAs but not in the vaccinated chickens without PTAs, indicating that H9N2-specific MDAs were indeed one of the reasons for H9N2 inactivated vaccine failure in the field. When different titers of PTAs were used to mimic MDAs in SPF chickens, high (HI = 12 log2) and medium (HI = log 9 log2) titers of PTAs reduced the generation of H9N2-specific antibodies after the first vaccination, but a booster dose would induce a high and faster humoral immune response even of PTA interference. This study strongly suggested that high or medium titers of MDAs might explain H9N2 inactivated vaccine failure in the field.

Gross pathology of high pathogenicity avian influenza virus H5N1 2021–2022 epizootic in naturally infected birds in the United Kingdom

High pathogenicity avian influenza virus (HPAIV) clade 2.3.4.4b has re-emerged in the United Kingdom in 2021–2022 winter season, with over 90 cases of HPAIV detected among poultry and captive birds in England, Scotland, Wales, and Northern Ireland. Globally, HPAIV H5N1 has also had a wide geographical dispersion, causing outbreaks in Europe, North America, Asia, and Africa, impacting on socioeconomic and wildlife conservation. It is important to raise awareness of the gross pathological features of HPAIV and subsequently aid disease investigation through definition of pathological indicators following natural infection. In this study, we report on the gross pathology of HPAI H5N1 in poultry species (chicken, turkey, pheasant, guineafowl, duck, goose), and captive or wild birds (mute swan, tufted duck, jackdaw, peahen, white-tailed eagle) that tested positive between October 2021 and February 2022. Pancreatic and splenic necrosis were the common pathological findings in both Galliformes and Anseriformes. In addition to the more severe lesions documented in Galliformes, we also noted increased detection of pathological changes in a broader range of Anseriformes particularly in domestic ducks, in contrast to those reported in previous seasons with other H5Nx HPAIV subtypes. A continual effort to characterise the pathological impact of the disease is necessary to update on the presentation of HPAIV for both domestic/captive and wild birds whilst guiding early presumptive diagnosis.