Highly Pathogenic Avian Influenza H5N1 Research Articles

Efficacy of Two Licensed Avian Influenza H5 Vaccines Against Challenge with a 2015 U.S. H5N2 clade 2.3.4.4 Highly Pathogenic Avian Influenza Virus in Domestic Ducks.

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses from the H5 goose/Guangdong lineage caused a major outbreak in poultry in the United States in 2015. Although the outbreak was controlled, vaccines were considered as an alternative control method, and new vaccines were approved and purchased by the U.S. Department of Agriculture National Veterinary Stockpile for emergency use. In this study, we evaluated the efficacy of two of these vaccines in protecting Pekin ducks (Anas platyrhynchos var. domestica) against challenge with a H5N2 HPAI poultry isolate. A recombinant alphavirus-based vaccine and an inactivated adjuvanted reverse genetics vaccine, both expressing the hemagglutinin gene of a U.S. H5 clade 2.3.4.4 isolate (A/Gyrfalcon/Washington/41088-6/2014 H5N8), were used to immunize the ducks. The vaccines were given either as single vaccination at 2 days of age or in a prime-boost strategy at 2 and 15 days of age. At 32 days of age, all ducks were challenged with A/turkey/Minnesota/12582/15 H5N2 HPAI virus clade 2.3.4.4. All ducks from the nonvaccinated challenge control group became infected and shed virus; one duck in this group presented mild ataxia, and a second duck died. No mortality or clinical signs were observed in vaccinated and challenged ducks, with the exception of one duck presenting with mild ataxia. Both vaccines, regardless of the vaccination strategy used, were immunogenic in ducks and reduced or prevented virus shedding after challenge. In conclusion, good protection against H5Nx infection was achieved in ducks vaccinated with the vaccines examined, which were homologous to the challenge virus, with prime-boost strategies conferring the best protection against infection.

Protective efficacy of an inactivated chimeric H7/H5 avian influenza vaccine against highly pathogenic avian influenza H7N9 and clade 2.3.4.4 H5 viruses

The highly pathogenic avian influenza (HPAI) H5 and H7N9 viruses pose a serious challenge to public health and the poultry industry in China. In this study, we generated a chimeric H7/H5 recombinant virus that expressed the entire HA1 region of the HPAI A/chicken/Guangdong/RZ/2017(H7N9) virus and the HA2 region of the HPAI A/chicken/Fujian/5/2016(H5N6) viruses. The resulting chimeric PR8-H7/H5 virus exhibited similar growth kinetics as the parental PR8-H5 and PR8-H7 viruses in vitro. The inactivated chimeric PR8-H7/H5 vaccine induced specific, cross-reactive hemagglutination inhibition antibodies against the H7 virus only but induced serum-neutralizing antibodies against both H7 and H5 viruses. Furthermore, the inactivated chimeric PR8-H7/H5 vaccine significantly reduced virus shedding and protected chickens from challenge with the HPAI H5N6 and H7N9 viruses. Our results suggested that the inactivated chimeric PR8-H7/H5 vaccine was effective against HPAI H5 and H7N9 viruses in chickens.

Tropism, replication competence, and innate immune responses of influenza virus: an analysis of human airway organoids and ex-vivo bronchus cultures

Human airway organoids are three-dimensional cultures derived from stem cells, which self-organise in ex-vivo conditions to form so-called mini-airways. The cellular morphology of these cultures is physiologically similar to the human airway, with cilia, goblet cells, and club cells facing the inner lumen and basal cells situated at the outer layer. The aim of this study was to compare replication competence, tissue tropism, and host responses elicited by human and avian strains of influenza A virus in ex-vivo human bronchus and human airway organoids. Between Sept 29, 2016, and Jan 4, 2017, we obtained ex-vivo cultures of the human bronchus and cultured human airway organoids from lung stem cells obtained from human lung tissues removed as part of the routine clinical care of patients undergoing surgical resection at the Department of Cardiothoracic Surgery, University of Hong Kong, Queen Mary Hospital, Hong Kong. We compared viral replication competence, tissue tropism, and cytokine and chemokine induction of avian influenza A viruses isolated from humans (Sh2/H7N9, H5N1/483, H5N6/39715), and human H1N1pdm/415742 in airway organoids and ex-vivo bronchus explant cultures. Virus tropism and replication kinetics of human and avian influenza A viruses in human airway organoids mimicked those found in ex-vivo cultures of human bronchus explants. In both airway organoids and bronchus explants, influenza A H1N1 subtype (H1N1) and avian influenza A H7N9 viruses replicated to significantly higher titres than did the highly pathogenic avian influenza (HPAI) H5N1, whereas HPAI H5N6 replication was moderate. H1N1, H7N9, and H5N6 viruses infected ciliated cells and goblet cells, but not basal cells in both airway organoids and bronchus explants. The expression of cytokines, interleukin 6, and interferon β, and the chemokine regulated-on-activation, normal T-cell expressed and secreted, was significantly higher in human airway organoids infected with HPAI H5N1 virus than H1N1pdm/415742, Sh2/H7N9, and H5N6/39715 viruses, and the expression of monocyte chemoattractant protein-1 was significantly higher in human organoids infected with HPAI H5N1 virus than H1N1pdm/415742 and Sh2/H7N9 viruses. Human airway organoid cultures provided results that were comparable to those observed in human ex-vivo bronchus cultures, and thus provide an alternative physiologically relevant experimental model for investigating virus tropism and replication competence that could be used to assess the pandemic threat of animal influenza viruses. US National Institute of Allergy and Infectious Diseases, Research Grants Council of the Hong Kong Special Administrative Region.

Mitigation strategies to reduce the generation and transmission of airborne highly pathogenic avian influenza virus particles during processing of infected poultry.

Airborne transmission of H5N1 highly pathogenic avian influenza (HPAI) viruses has occurred among poultry and from poultry to humans during home or live-poultry market slaughter of infected poultry, and such transmission has been experimentally reproduced. In this study, we investigated simple, practical changes in the processing of H5N1 virus-infected chickens to reduce infectious airborne particles and their transmission. Our findings suggest that containing the birds during the killing and bleeding first step by using a disposable plastic bag, a commonly available cooking pot widely used in Egypt (halla), or a bucket significantly reduces generation of infectious airborne particles and transmission to ferrets. Similarly, lack of infectious airborne particles was observed when processing vaccinated chickens that had been challenged with HPAI virus. Moreover, the use of a mechanical defeatherer significantly increased total number of particles in the air compared to manual defeathering. This study confirms that simple changes in poultry processing can efficiently mitigate generation of infectious airborne particles and their transmission to humans.

Multi-clade H5N1 virus-like particles: Immunogenicity and protection against H5N1 virus and effects of beta-propiolactone

During the past decade, H5N1 highly pathogenic avian influenza (HPAI) viruses have diversified genetically and antigenically, suggesting the need for multiple H5N1 vaccines. However, preparation of multiple vaccines from live H5N1 HPAI viruses is difficult and economically not feasible representing a challenge for pandemic preparedness. Here we evaluated a novel multi-clade recombinant H5N1 virus-like particle (VLP) design, in which H5 hemagglutinins (HA) and N1 neuraminidase (NA) derived from four distinct clades of H5N1 virus were co-localized within the VLP structure. The multi-clade H5N1 VLPs were prepared by using a recombinant baculovirus expression system and evaluated for functional hemagglutination and neuraminidase enzyme activities, particle size and morphology, as well as for the presence of baculovirus in the purified VLP preparations. To remove residual baculovirus, VLP preparations were treated with beta-propiolactone (BPL). Immunogenicity and efficacy of multi-clade H5N1 VLPs were determined in an experimental ferret H5N1 HPAI challenge model, to ascertain the effect of BPL on immunogenicity and protective efficacy against lethal challenge. Although treatment with BPL reduced immunogenicity of VLPs, all vaccinated ferrets were protected from lethal challenge with influenza A/VietNam/1203/2004 (H5N1) HPAI virus, indicating that multi-clade VLP preparations treated with BPL represent a potential approach for pandemic preparedness vaccines.

Characterization of highly pathogenic avian influenza H5N8 virus from Egyptian domestic waterfowl in 2017

ABSTRACTIn 2016, the highly pathogenic avian influenza (HPAI) H5N8 virus was detected in wild birds for the first time in Egypt. In the present study, we identified the HPAI virus H5N8 of clade 2.3.4.4 from domestic waterfowl in Egypt, suggesting its transmission to the domestic poultry from the migratory birds. Based on partial haemagglutinin gene sequence, this virus has a close genetic relationship with subtype H5N8 viruses circulating in Asia and Europe. Pathologically, H5N8 virus in hybrid duck induced nervous signs accompanied by encephalomalacia, haemorrhages, nonsuppurative encephalitis and nonsuppurative vasculitis. The granular layer of cerebellum showed multifocal areas of hydropic degeneration and the Purkinje cell neurons were necrotized or lost. Additionally, the lung, kidney and spleen were congested, and necrotizing pancreatitis was also observed. The co-circulation of both HPAI H5N1 and H5N8 subtypes with the low pathogenic avian influenza H9N2 subtype complicate the control of avian influenza in Egypt with the possibility of emergence of new reassortant viruses. Therefore, continuous monitoring with implementation of strict control measures is required.Research highlightsHPAI H5N8 virus clade 2.3.4.4 was detected in domestic ducks and geese in Egypt in 2017.Phylogenetically, the virus was closely related to HPAI H5N8 viruses identified in Asia and EuropeNonsuppurative encephalitis was widely observed in HPAI H5N8 virus-infected ducks.Degeneration of the cerebellar granular layer was found in most of the brain tissues examined.

Evolutionary dynamics of avian influenza A H7N9 virus across five waves in mainland China, 2013–2017

Since its emergence in March 2013, novel avian influenza A H7N9 virus has triggered five epidemics of human infections in China. This raises concerns about the pandemic threat of this quickly evolving H7N9 subtype for humans. In this study, we evaluated all available genomes for H7N9 and H9N2 influenza A viruses. Our assessment discovered that H7N9 of the 1st wave had the lowest nucleotide diversity, which then experienced substantial and rapid population expansion from a small founder population. From the 2nd wave, their nucleotide diversity increased quickly, indicating that H7N9 viruses had acquired larger populations and mutations after their initial emergence in 2013. After the phylogeographic divergence in the 2nd wave, although the HA and NA genes from different regions differed, compared to previous epidemics, the evolving H7N9 viruses in the 5th wave lost most of their previous clades. The highly pathogenic avian influenza (HPAI) H7N9 viruses in the 5th wave clustered together, and clustered close to the low pathogenic avian influenza (LPAI) virus isolated from the Pearl River Delta in the 3rd and 4th waves. This result supports the origin of HPAI H7N9 viruses was in the Pearl River Delta. In the 5th wave, although both HPAI and LPAI H7N9 viruses were isolated from the Pearl River Delta, their HA and NA genes were phylogenetically distinct.

Experimental infection of H5N1 and H5N8 highly pathogenic avian influenza viruses in Northern Pintail (Anas acuta).

The wide geographic spread of Eurasian Goose/Guangdong lineage highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses by wild birds is of great concern. In December 2014, an H5N8 HPAI clade 2.3.4.4 Group A (2.3.4.4A) virus was introduced to North America. Long-distance migratory wild aquatic birds between East Asia and North America, such as Northern Pintail (Anas acuta), were strongly suspected of being a source of intercontinental transmission. In this study, we evaluated the pathogenicity, infectivity and transmissibility of an H5N8 HPAI clade 2.3.4.4A virus in Northern Pintails and compared the results to that of an H5N1 HPAI clade 2.3.2.1 virus. All of Northern Pintails infected with either H5N1 or H5N8 virus lacked clinical signs and mortality, but the H5N8 clade 2.3.4.4 virus was more efficient at replicating within and transmitting between Northern Pintails than the H5N1 clade 2.3.2.1 virus. The H5N8-infected birds shed high titre of viruses from oropharynx and cloaca, which in the field supported virus transmission and spread. This study highlights the role of wild waterfowl in the intercontinental spread of some HPAI viruses. Migratory aquatic birds should be carefully monitored for the early detection of H5 clade 2.3.4.4 and other HPAI viruses.

Phylogeography of H5N1 avian influenza virus in Indonesia.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are a major concern to human and animal health in Indonesia. This study aimed to characterize transmission dynamics of H5N1 over time using novel Bayesian phylogeography methods to identify factors which have influenced the spread of H5N1 in Indonesia. We used publicly available hemagglutinin sequence data sampled between 2003 and 2016 to model ancestral state reconstruction of HPAI H5N1 evolution. We found strong support for H5N1 transmission routes between provinces in Java Island and inter-island transmissions, such as between Nusa Tenggara and Kalimantan Islands, not previously described. The spread is consistent with wild bird flyways and poultry trading routes. H5N1 migration was associated with the regions of high chicken densities and low human development indices. These results can be used toinform more targeted planning of H5N1 control and prevention activities in Indonesia.

Risk factors associated with highly pathogenic avian influenza subtype H5N8 outbreaks on broiler duck farms in South Korea.

Highly Pathogenic Avian Influenza (HPAI) subtype H5N8 outbreaks occurred in poultry farms in South Korea in 2014 resulting in significant damage to the poultry industry. Between 2014 and 2016, the pandemic disease caused significant economic loss and social disruption. To evaluate the risk factors for HPAI infection in broiler duck farms, we conducted a retrospective case-control study on broiler duck farms. Forty-three farms with confirmed laboratories on premises were selected as the case group, and 43 HPAI-negative farms were designated as the control group. Control farms were matched based on farm location and were within a 3-km radius from the case premises. Spatial and environmental factors were characterized by site visit and plotted through a geographic information system (GIS). Univariable and multivariable logistic regression models were developed to assess possible risk factors associated with HPAI broiler duck farm infection. Four final variables were identified as risk factors in a final multivariable logistic model: "Farms with ≥7 flocks" (odds ratio [OR]=6.99, 95% confidence interval [CI] 1.34-37.04), "Farm owner with ≥15years of raising poultry career" (OR=7.91, 95% CI 1.69-37.14), "Presence of any poultry farms located within 500m of the farm" (OR=6.30, 95% CI 1.08-36.93) and "Not using a faecal removal service" (OR=27.78, 95% CI 3.89-198.80). This highlights that the HPAI H5N8 outbreaks in South Korea were associated with farm owner education, number of flocks and facilities and farm biosecurity. Awareness of these factors may help to reduce the spread of HPAI H5N8 across broiler duck farms in Korea during epidemics. Greater understanding of the risk factors for H5N8 may improve farm vulnerability to HPAI and other subtypes and help to establish policies to prevent re-occurrence. These findings are relevant to global prevention recommendations and intervention protocols.

Wild ducks excrete highly pathogenic avian influenza virus H5N8 (2014–2015) without clinical or pathological evidence of disease

Highly pathogenic avian influenza (HPAI) is essentially a poultry disease. Wild birds have traditionally not been involved in its spread, but the epidemiology of HPAI has changed in recent years. After its emergence in southeastern Asia in 1996, H5 HPAI virus of the Goose/Guangdong lineage has evolved into several sub-lineages, some of which have spread over thousands of kilometers via long-distance migration of wild waterbirds. In order to determine whether the virus is adapting to wild waterbirds, we experimentally inoculated the HPAI H5N8 virus clade 2.3.4.4 group A from 2014 into four key waterbird species—Eurasian wigeon (Anas penelope), common teal (Anas crecca), mallard (Anas platyrhynchos), and common pochard (Aythya ferina)—and compared virus excretion and disease severity with historical data of the HPAI H5N1 virus infection from 2005 in the same four species. Our results showed that excretion was highest in Eurasian wigeons for the 2014 virus, whereas excretion was highest in common pochards and mallards for the 2005 virus. The 2014 virus infection was subclinical in all four waterbird species, while the 2005 virus caused clinical disease and pathological changes in over 50% of the common pochards. In chickens, the 2014 virus infection caused systemic disease and high mortality, similar to the 2005 virus. In conclusion, the evidence was strongest for Eurasian wigeons as long-distance vectors for HPAI H5N8 virus from 2014. The implications of the switch in species-specific virus excretion and decreased disease severity may be that the HPAI H5 virus more easily spreads in the wild-waterbird population.

Comparative Epidemiology of Highly Pathogenic Avian Influenza Virus H5N1 and H5N6 in Vietnamese Live Bird Markets: Spatiotemporal Patterns of Distribution and Risk Factors.

Highly pathogenic avian influenza (HPAI) H5N1 virus has been circulating in Vietnam since 2003, whilst outbreaks of HPAI H5N6 virus are more recent, having only been reported since 2014. Although the spatial distribution of H5N1 outbreaks and risk factors for virus occurrence has been extensively studied, there have been no comparative studies for H5N6. Data collected through active surveillance of Vietnamese live bird markets (LBMs) between 2011 and 2015 were used to explore and compare the spatiotemporal distributions of H5N1- and H5N6-positive LBMs. Conditional autoregressive models were developed to quantify spatiotemporal associations between agroecological factors and the two HPAI strains using the same set of predictor variables. Unlike H5N1, which exhibited a strong north–south divide, with repeated occurrence in the extreme south of a cluster of high-risk provinces, H5N6 was homogeneously distributed throughout Vietnam. Similarly, different agroecological factors were associated with each strain. Sample collection in the months of January and February and higher average maximum temperature were associated with higher likelihood of H5N1-positive market-day status. The likelihood of market days being positive for H5N6 increased with decreased river density, and with successive Rounds of data collection. This study highlights marked differences in spatial patterns and risk factors for H5N1 and H5N6 in Vietnam, suggesting the need for tailored surveillance and control approaches.

Comparison of the pathogenic potential of highly pathogenic avian influenza (HPAI) H5N6, and H5N8 viruses isolated in South Korea during the 2016–2017 winter season

Highly pathogenic avian influenza (HPAI) A(H5N6) and A(H5N8) virus infections resulted in the culling of more than 37 million poultry in the Republic of Korea during the 2016/17 winter season. Here we characterize two representative viruses, A/Environment/Korea/W541/2016 [Em/W541(H5N6)] and A/Common Teal/Korea/W555/2017 [CT/W555(H5N8)], and evaluate their zoonotic potential in various animal models. Both Em/W541(H5N6) and CT /W555(H5N8) are novel reassortants derived from various gene pools of wild bird viruses present in migratory waterfowl arising from eastern China. Despite strong preferential binding to avian virus–type receptors, the viruses were able to grow in human respiratory tract tissues. Em/W541(H5N6) was found to be highly pathogenic in both chickens and ducks, while CT/W555(H5N8) caused lethal infections in chickens but did not induce remarkable clinical illness in ducks. In mice, both viruses appeared to be moderately pathogenic and displayed limited tissue tropism relative to HPAI H5N1 viruses. Em/W541(H5N6) replicated to moderate levels in the upper respiratory tract of ferrets and was detected in the lungs, brain, spleen, liver, and colon. Unexpectedly, two of three ferrets in direct contact with Em/W541(H5N6)-infected animals shed virus and seroconverted at 14 dpi. CT/W555(H5N8) was less pathogenic than the H5N6 virus in ferrets and no transmission was detected. Given the co-circulation of different, phenotypically distinct, subtypes of HPAI H5Nx viruses for the first time in South Korea, detailed virologic investigations are imperative given the capacity of these viruses to evolve and cause human infections.

A two dose immunization with an inactivated reassortant H5N2 virus protects chickens against lethal challenge with homologous 2.3.2.1 clade and heterologous 2.2 clade highly pathogenic avian influenza H5N1 viruses

The present study was aimed at generating a reassortant vaccine candidate virus with clade 2.3.2.1 Hemagglutinin (HA) and its evaluation in a challenge study for protection against homologous (2.3.2.1 clade) and heterologous (2.2 clade) highly pathogenic avian influenza (HPAI) H5N1 viruses. Plasmid-based reverse genetics technique was used to rescue a 5 + 3 reassortant H5N2 strain containing the modified HA of H5N1 (clade 2.3.2.1), the Neuraminidase (NA) of H9N2, the Matrix (M) of H5N1 and the internal genes of A/WSN/33 H1N1. In addition, another 6 + 2 reassortant virus containing modified HA from H5N1 (clade 2.3.2.1), the NA from H9N2 and the internal genes of A/WSN/33 H1N1 was also rescued. The 5 + 3 reassortant H5N2 virus could grow to a higher titer in both MDCK cells and chicken eggs compared to the 6 + 2 reassortant H5N2 virus. The vaccine containing the inactivated 5 + 3 reassortant H5N2 virus was used in a two-dose immunization regime which protected specific pathogen free (SPF) chickens against two repeated challenges with homologous 2.3.2.1 clade and heterologous 2.2 clade HPAI H5N1 viruses. The 5 + 3 reassortant H5N2 virus based on clade 2.3.2.1 generated in this study can be effective in protecting chickens in the case of an outbreak caused by antigenically different clade 2.2 HPAI H5N1 viruses and opens the way to explore its applicability as potential vaccine candidate especially in the Asian countries reporting these clades frequently. The study also indicates that sequential immunization can broaden protection level against antigenically diverse strains of H5N1 viruses.

Study of an Outbreak of Highly Pathogenic Avian Influenza H5N8 in Commercial Pekin Ducks (Anas platyrhynchos domesticus) in California

A February 2015 outbreak of highly pathogenic avian influenza (HPAI) H5N8 in a flock of commercial Pekin ducks ( Anas platyrhynchos domesticus) in California was investigated in detail. Approximately 17,349 five-wk-old ducks experienced an increased mortality from a normal of eight birds per day to 24, 18, 24, 33, and 61 birds per day, respectively, in the last 5 days prior to flock depopulation. Clinically, there was decreased water and feed consumption, and approximately 1.0% of the affected flock exhibited neurologic signs. Necropsy of five clinically ill ducks revealed pale, patchy areas on the epicardium in two birds, pale foci of necrosis in the liver of one bird, and airsacculitis in three birds. Histopathology revealed multifocal nonsuppurative encephalomyelitis, myocarditis, myositis, pancreatitis, hepatitis, and glossitis. Immunohistochemistry revealed avian influenza virus (AIV) nucleoprotein in the nucleus and cytoplasm of various cells in the aforementioned organs, as well as in the skin and feathers. Eight of the 10 sera samples tested were positive for avian influenza antibodies by agar gel immunodiffusion serology. Oropharyngeal and cloacal swabs taken from 15 birds, as well as from the lungs, livers, pancreas, and spleen, were positive for AIV by real-time reverse transcriptase (rRT) PCR. AIV was isolated and typed as Eurasian lineage HPAI H5N8, clade 2.3.4.4, by the National Veterinary Services Laboratory, Ames, IA. Extensive surveillance of birds for AIV around the 10-km zone did not reveal any additional cases. Ducks on the affected premises were humanely euthanatized by foam and composted in-house, the houses were heated to 57 C for 4 days, and swabs were taken periodically from the compost to ensure negativity for AIV by rRT-PCR. The compost and litter were then removed, and the house was pressure cleaned, disinfected, and repopulated approximately 120 days after euthanatization of the ducks.

Global spatial risk pattern of highly pathogenic avian influenza H5N1 virus in wild birds: A knowledge-fusion based approach

Highly pathogenic avian influenza (HPAI) H5N1 viruses have continuously circulated throughout much of the world since 2003, resulting in huge economic losses and major public health problems. Wild birds have played an important role in the spread of H5N1 HPAI. To understand its spatial distribution, H5N1 HPAI have been studied by many disciplines from different perspectives, but only one kind of disciplinary knowledge was involved, which has provided limited progress in understanding. Combining risk information from different disciplines based on knowledge fusion can provide more accurate and detailed information. In this study, local k function, phylogenetic tree analysis, and logistic spatial autoregressive models were used to explore the global spatial pattern of H5N1 HPAI based on outbreak data in wild birds, genetic sequences, and risk factors, respectively. On this basis, Dempster-Shafer (D-S) evidence theory was further applied to study the spatial distribution of H5N1 HPAI. We found D-S evidence theory was more robust and reliable than the other three methods, providing technical and methodological support for application to the research of other diseases. The shortest distance to wild bird migration routes, roads and railways, elevation, the normalized difference vegetation index (NDVI), land use and land cover (LULC) and infant mortality rates (IMR) were significantly associated with the occurrence of H5N1 HPAI. The high-risk areas were mainly located in Northern and Central Europe, the eastern Mediterranean, and East and Southeast Asia. High-risk clusters were closely related to the social, economic and ecological environment of the region. Locations where the potential transmission risk remains high should be prioritized for control efforts.

220 mutation in the hemagglutinin of avian influenza A (H7N9) virus alters antigenicity during vaccine strain development

ABSTRACTSince the first confirmed case of H7N9 infection was reported in China, there have been five epidemic waves of human H7N9 infections between 2013 and 2017. The fifth wave differed from the previous four waves in that highly pathogenic avian influenza (HPAI) H7N9 viruses with multiple basic amino acids at the cleavage site were detected in humans, poultry and environmental samples. The HPAI H7N9 viruses were genetically and antigenically distinct from previous H7N9 viruses. Therefore, a new candidate vaccine virus(CVV) derived from a HPAI A/Guangdong/17SF003/2016-like virus was proposed by the World Health Organization(WHO). According to the WHO recommendations, we constructed a new CVV using reverse genetic technology, with a (6+2) gene constitution. The (6+2) reassortant virus possessed hemagglutinin(HA) with multiple basic amino acids removed and the neuraminidase from A/Guangdong/SF003/2016 in a high-yield A/Puerto Rico/8/34 virus backbone. Sequence analysis confirmed that no mutations had occurred in the HA of V1E1(the initial CVV rescued in Vero cells and followed by passage in eggs), but a mixture of arginine (R)/glycine (G)/isoleucine (I) was detected at position 220 (H3 numbering) in the HA of V1E2 to V1E5 with different percentages. Furthermore, V1E5 showed improved growth characteristics and immunogenicity compared with V1E1, and retained low pathogenicity in chickens and chicken embryos, but the mutation changed its antigenicity. Our study indicates that antigenic changes should be closely monitored during the development of H7N9 CVV in eggs. Additionally, although V1E5 changes the antigenicity, the antisera had some reactivity to previous H7N9 CVVs.

An Egyptian HPAI H5N1 isolate from clade 2.2.1.2 is highly pathogenic in an experimentally infected domestic duck breed (Sudani duck).

The highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause major problems in poultry and can, although rarely, cause human infection. Being enzootic in domestic poultry, Egyptian isolates are continuously evolving, and novel clades vary in their pathogenicity in avian hosts. Considering the importance of domestic ducks as natural hosts of HPAI H5N1 viruses and their likelihood of physical contact with other avian hosts and humans, it is of utmost importance to characterize the pathogenicity of newly emerged HPAI strains in the domestic duck. The most recently identified Egyptian clade 2.2.1.2 HPAI H5N1 viruses have been isolated from naturally infected pigeons, turkeys and humans. However, essentially nothing is known about their pathogenicity in domestic ducks. We therefore characterized the pathogenicity of an Egyptian HPAI H5N1 isolate A/chicken/Faquos/amn12/2011 (clade 2.2.1.2) in Sudani duck, a domestic duck breed commonly reared in Egypt. While viral transcription (HA mRNA) was highest in lung, heart and kidney peaking between 40 and 48hpi, lower levels were detected in brain. Weight loss of infected ducks started at 16hpi and persisted until 120hpi. The first severe clinical signs were noted by 32hpi and peaked in severity at 72 and 96hpi. Haematological analyses showed a decline in total leucocytes, granulocytes, platelets and granulocyte/lymphocyte ratio, but lymphocytosis. Upon necropsy, lesions were obvious in heart, liver, spleen and pancreas and consisted mainly of necrosis and petechial haemorrhage. Histologically, lungs were the most severely affected organs, whereas brain only showed mild neuronal degeneration and gliosis at 48hpi despite obvious neurological clinical signs. Taken together, our results provide first evidence that this HPAI H5N1 isolate (clade 2.2.1.2) is highly pathogenic to Sudani ducks and highlight the importance of this breed as potential reservoir and disseminator of HPAI strains from this clade.

Spatial and temporal analyses of highly pathogenic avian influenza H5N1 re-emergence in Nigeria, 2015

Introduction : highly pathogenic avian influenza (HPAI) virus H5N1 kills up to 90 - 100% of domestic fowl flock during outbreaks globally. This study, conducted from January 8, 2015 to August 6, 2015, aimed to assess the space-time distribution pattern of the re-emergence of HPAI H5N1 in Nigeria. Methods : secondary analysis was done on confirmed cases of HPAI data abstracted from records of the Federal Department of Veterinary and Pest Control Service, Abuja. We sorted cases into confirmed and suspected HPAI. Geographic coordinates of affected farms was obtained using Google Earth Pros. We conducted analysis of the data with Kulldorff’s two-dimensional spatial scan statistics based on the use of circular window to identify whether the HPAI cases were randomly distributed over space or not. Spatial scan was used to detect clusters in space (assuming a Bernoulli distribution). Primary and secondary clusters were determined using likelihood-ratio test statistics. Time and space-time permutation was developed for prediction of possible outbreak. Chi-square was calculated at significant level set at 5%. Results : the virus affected 20 states (55.6%) in Nigeria. HPAI H5N1 epidemics occurred in all the six geo-political zones of Nigeria. Primary cluster and two secondary clusters of purely spatial scan statistics were all significant at p ≤ 0.05, and revealed that 95.1% of H5N1-infected farms were located within a 0.091km radius (Log likelihood ratio = 25.68). The centre coordinates for the clusters were found to be areas of high human density and considerable swine production. The Space-time permutation model identified a significant prospective outbreaks within 3.5 years interval in Plateau State (χ2 = 58.08, P Conclusion : this finding portends significant potential for co-infection and re-assortment of the virus with the human and swine strains. We recommend the need for enhanced surveillance in both poultry and swine populations in Nigeria.

Effects of Highly Pathogenic Avian Influenza H5N1 Outbreak in Nepal from Financial and Social Perspectives: A Case Study

The outbreaks of highly pathogenic avian influenza (HPAI) H5N1 have serious public health, economic and social implications. After the first two report of HPAI H5N1 outbreak in Nepal in Jhapa in 2009, a third outbreak was reported in Pokhara the following year where more than 11,000 birds were culled to control the disease. However, the financial and social effects of the Pokhara bird flu outbreak were never assessed. The objectives of this study were to evaluate the local financial and social effects of HPAI H5N1 outbreak in Pokhara and to understand farmers and consumers perceptions on bird flu and the control measures implemented by the government. A total of 200 people including poultry farmers, butchers, veterinary drug dealers, feed suppliers, and consumers were interviewed using the semi-structured questionnaire. The result showed that the direct loss of Pokhara bird flu outbreak from the culling of birds was more than 4.5 million Nepali rupees. The small scale commercial and backyard farmers and the butchers were the most affected from this outbreak. A large proportion of the consumers temporarily stopped to consume chicken meat and shifted to other meat sources. Most of the participants in this study had at least heard about the bird flu. The farmers were unhappy by the compensation provided by the government and perceived that the government control efforts were not satisfactory and were provided little assistance for their rehabilitation. In conclusion, there was a momentary local financial and social effect of the Pokhara bird flu outbreak which may not very important from the macroeconomic point of view but is important for the affected small-scale farmers and the butchers. It is recommended that the government review their policy and provide small scale commercial and backyard farmer a compensation that is closer to the market price and promote largescale poultry farmers to go for insurance.